Building and sequencing method of single-stranded nucleic acid cyclic library

Abstract

Belongs to the field of gene sequencing, and particularly relates to a library building and sequencing method of a single-stranded nucleic acid cyclic library. According to the method, a sequencing primer binding sequence is combined in a linker molecule or the sequencing primer binding sequence is respectively contained in the linker molecule and an extension reaction primer, so that the reaction step for connecting a sequencing linker is reduced, the loss of a nucleic acid template is reduced, the linker connecting efficiency is improved, and the yield of the sequencing linker is improved. The utilization rate of an original nucleic acid template is increased, and rapid library building and sequencing of various nucleic acid samples are realized.

Description

Construction and sequencing methods of single-stranded nucleic acid circular libraries Technical Field

[0001] The present invention belongs to the field of gene sequencing, and more specifically, relates to a method for constructing and sequencing a single-stranded nucleic acid circular library. Background Art

[0002] Currently, DNA sequencing libraries are typically constructed using intact genomic DNA with sufficient starting volume. Conventional double-stranded DNA is typically used for library construction. The general steps include fragmenting the DNA into fragments of the desired size, performing end-repair, and then ligating double-stranded adapters to the ends of the DNA fragment templates via a simple ligase reaction to obtain the library required for sequencing. However, for some special samples, such as formalin-fixed (FFPE) and paraffin-embedded biological tissue samples, forensic analysis samples, and paleontological fossils, the DNA extracted from these samples is often severely degraded, and the amount of DNA extracted can be very low. These DNA molecules often contain both double-stranded and single-stranded DNA molecules, or single-stranded breaks can occur within double-stranded DNA molecules. Furthermore, in methylation sequencing, DNA undergoes severe damage after bisulfite conversion, resulting in the loss of most DNA molecules and the fragmentation of DNA into short fragments that remain single-stranded. Furthermore, extracellular free DNA in plasma or other body fluid samples is itself short DNA fragments, and the DNA content is also very low.

[0003] For these special samples, the common double-stranded adapter ligation library construction method is not suitable, because conventional library construction methods can only target complete double-stranded DNA molecules, and cannot or rarely add adapters to all DNA template molecules other than complete double-stranded DNA molecules, which will cause the loss of a large number of original DNA template molecules. Single-stranded library construction technology can utilize these single-stranded DNA template molecules, add adapters to these single-stranded DNA molecules, and construct libraries, thereby minimizing the loss of these irregular double-stranded DNA template molecules and maximizing the utilization rate of DNA template molecules. Therefore, the efficiency of library construction is improved, and the effective data volume and data quality of sequencing are improved. In addition, single-stranded library construction technology can also better use the above special samples for corresponding application detection, such as liquid biopsy for extracellular free DNA (cfDNA) methylation sequencing, genomic research on paleontological DNA, forensic identification, and tumor detection of FFPE samples.

[0004] Existing single-strand library construction technologies mainly include traditional single-strand library construction methods, Swift Adaptase single-strand library construction technology, random primer plus adapter method, terminal transferase plus adapter method and SPALT (Splinted adaptor tagging) technology.

[0005] Traditional single-stranded library construction methods involve directly ligating single-stranded sequencing adapters to single-stranded DNA template molecules using T4 RNA ligase. Typically, T4 RNA ligase 2 (truncated) is used to catalyze the ligation of pre-adenylated 5'-end DNA adapters to the 3' end of the single-stranded DNA template. The 3' end of the adapter is blocked to reduce the formation of adapter dimers. After purification, the resulting product is then added with a 5'-end adapter and T4 RNA ligase. The 5'-end adapter is then ligated to the single-stranded DNA template to which the 3'-end adapter has been attached. After both ends have been attached, PCR amplification is performed. If a truncated adapter was previously attached, PCR can also complete the remaining adapter.

[0006] Swift's Adaptase single-stranded library construction technology first uses single-stranded ligase to connect a universal sequencing adapter with a random primer to the 3' end of the single-stranded DNA template, and then uses a complementary primer to the 3' end adapter primer binding sequence for amplification to convert the single-stranded DNA template into a double-stranded DNA template. Then, through a conventional adapter ligation reaction, a 5' end sequencing adapter is added to the 5' end of the newly synthesized chain to complete the library construction.

[0007] The random primer single-strand library construction technology uses 6nt to 8nt random primers to combine with the single-stranded DNA template for extension, thereby synthesizing a double strand, and then adding a 5' end sequencing adapter to the 5' end of the newly synthesized chain to complete the library construction.

[0008] The single-stranded library construction technology using terminal transferase is similar to the single-stranded library construction technology using random primers. First, terminal transferase is used to add continuous bases to the 3' end of the single-stranded DNA template. Then, the continuous sequence is used as the primer binding position for extension to convert the single-stranded DNA template into a double-stranded DNA. Then, a 5' sequencing adapter is added to the 5' end of the newly synthesized chain to complete the library construction.

[0009] SPALT (Splinted Adaptor Tagging) technology involves adding a double-stranded adapter containing a stretch of random bases to the 3' end of the single-stranded DNA template. The double-stranded adapter contains a stretch of random bases that complement the single-stranded DNA. A second double-stranded adapter containing random bases is then added to the 5' end of the single-stranded template DNA. After adding adapters to both ends of the single-stranded DNA, PCR amplification is performed to complete the construction of the single-stranded library.

[0010] In addition, if the above methods are to eventually construct a single-stranded circular library, the single strands need to be further denatured, and then circularized and connected using DNA ligase and oligonucleotide chains with complementary linkers at both ends.

[0011] As can be seen from the above, the traditional single-stranded library construction method uses T4 RNA ligase 2 (truncated type), which requires a DNA adapter modified by 5' pre-adenylation at the end, wherein the price of 5' pre-adenylation at the end is expensive, and difficult to synthesize, and only a very small number of manufacturers can produce, resulting in high cost. Moreover, it is necessary to connect adapters at both ends of the DNA template, and to perform multiple purifications, resulting in low template utilization and long library construction time. Adaptase single-stranded library construction technology, random primer plus adapter method, terminal transferase plus adapter method and SPALT technology are also all respectively connected to the adapter of 3' end and 5' end at both ends of the DNA template, and only after adding adapters at both ends can PCR amplification be performed to amplify the template. These all increase the experimental steps and simultaneously result in the loss of the original DNA template, reducing the utilization of the DNA template.

[0012] Therefore, there is an urgent need in the art for a method for constructing a single-stranded nucleic acid circular library. This method does not require the addition of adapters to both ends of the single-stranded nucleic acid template, nor does it require the use of costly special end modifications. This reduces the number of reaction steps and reduces costs, reduces the loss of nucleic acid templates, increases the efficiency of adapter ligation, and improves the utilization rate of the original nucleic acid template.

[0013] Summary of the Invention

[0014] As described above, existing methods for constructing single-stranded nucleic acid circular libraries all require attaching adapters to both ends of the nucleic acid template. PCR amplification can only be performed after both ends have been attached, which increases the number of experimental steps and costs, while also resulting in loss of the original nucleic acid template and reducing the utilization rate of the nucleic acid template. Therefore, there is an urgent need in the art for a method for constructing a single-stranded nucleic acid circular library that does not require attaching adapters to both ends of the single-stranded nucleic acid template and does not require the use of costly special end modifications, thereby enabling rapid library construction and sequencing of a variety of nucleic acid samples, particularly special nucleic acid samples such as short nucleic acid fragments, extracellular free nucleic acid, and degraded nucleic acid.

[0015] Therefore, in a first aspect, the present invention provides a method for constructing a single-stranded nucleic acid circular library, comprising the following steps:

[0016] 1) Providing a single-stranded nucleic acid template;

[0017] 2) forming a partially double-stranded linker element between the linker molecule and the auxiliary chain, wherein the 5' end of the linker molecule is phosphorylated, the 3' end of the linker molecule is modified and blocked, the 5' end portion of the auxiliary chain is reverse complementary to the 5' end portion of the linker molecule, and the 3' end of the auxiliary chain has a random base sequence that is hybridized and complementary to the 3' end portion of the single-stranded nucleic acid template;

[0018] 3) ligating the linker molecule in the linker element to the 3' end of the single-stranded nucleic acid template using a ligase, thereby obtaining a nucleic acid fragment with a partial double strand;

[0019] 4) Performing a single-stranded circularization reaction on the product in step 3) to obtain a single-stranded nucleic acid circular library.

[0020] In a second aspect, the present invention further provides a method for constructing a single-stranded nucleic acid circular library, comprising the following steps:

[0021] 1) Providing a single-stranded nucleic acid template;

[0022] 2) forming a partially double-stranded linker element between the linker molecule and the auxiliary chain, wherein the 5' end of the linker molecule is phosphorylated, the 3' end portion of the auxiliary chain is reverse complementary to the 3' end portion of the linker molecule, and the 5' end of the auxiliary chain has a random base sequence that is hybridized and complementary to the 5' end portion of the single-stranded nucleic acid template;

[0023] 3) ligating the linker molecule in the linker element to the 5' end of the single-stranded nucleic acid template using a ligase, thereby obtaining a nucleic acid fragment with a partial double strand;

[0024] 4) Performing a single-stranded circularization reaction on the product obtained in step 3) to obtain a single-stranded nucleic acid circular library.

[0025] In a third aspect, the present invention further provides a method for sequencing single-stranded circular DNA, the method comprising:

[0026] a) performing the method for constructing a single-stranded nucleic acid circular library according to the first or second aspect of the present invention to obtain a single-stranded nucleic acid circular library;

[0027] b) performing an amplification reaction on the single-stranded nucleic acid circular library product obtained in step a) and sequencing to obtain sequencing data.

[0028] In a fourth aspect, the present invention provides a kit for constructing a single-stranded nucleic acid circular library, the kit comprising:

[0029] (1) an adapter molecule, for example, the adapter molecule comprises at least one sequencing primer binding sequence or a combination of multiple sequencing primer binding sequences;

[0030] (2) an auxiliary chain, wherein one end of the auxiliary chain has a sequence that is reverse complementary to a portion of the sequence at the connecting end of the linker molecule, and the other end of the auxiliary chain has a random base sequence that is complementary to the hybridization end of the single-stranded nucleic acid template;

[0031] (3) Ligase.

[0032] The beneficial effects of the present invention include one or more of the following:

[0033] 1) By combining multiple sequencing primer binding sequences in an adapter molecule or separately including multiple sequencing primer binding sequences in an adapter molecule and an extension reaction primer, the reaction steps for connecting sequencing adapters are reduced, the loss of nucleic acid templates is reduced, the efficiency of adapter connection is increased, and the utilization rate of the original nucleic acid template is improved;

[0034] 2) Only blocking modifications such as phosphorylation, C3-Spacer modification, amino modification, or C6 Spacer modification are required, thus reducing costs;

[0035] 3) Realize the rapid library construction and sequencing of various nucleic acid samples, especially special nucleic acid samples such as short nucleic acid fragments, extracellular free nucleic acids, and degraded nucleic acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other implementation plans can be obtained based on these drawings without paying any creative work.

[0037] Figure 1 is a schematic flow diagram of an embodiment of a method for rapid construction of a single-stranded DNA circular library. In the library construction method shown in this figure, the adapter molecule comprises a combination of two sequencing primer binding sequences, and the adapter molecule is connected to the 3' end of the single-stranded DNA template.

[0038] Figure 2 is a schematic flow chart of another embodiment of a method for rapidly constructing a single-stranded DNA circular library. In the library construction method shown in this figure, the adapter molecule only contains a sequencing primer binding sequence, and the adapter molecule is connected to the 3' end of the single-stranded DNA template.

[0039] FIG3 is a schematic diagram of the location of the tag sequence. The tag sequence can be located between multiple sequencing primer binding sequences in the adapter molecule, or the tag sequence can be located in the extension primer.

[0040] Figure 4 is a schematic flow diagram of an embodiment of a method for rapidly constructing a single-stranded DNA circular library. In the library construction method shown in this figure, the adapter molecule comprises a combination of two sequencing primer binding sequences, and the adapter molecule is connected to the 5' end of the single-stranded DNA template. DETAILED DESCRIPTION

[0041] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the embodiments described are only part of the embodiments of the present invention, rather than all the embodiments. All other embodiments that can be obtained by a person of ordinary skill in the art based on the embodiments of the present invention are within the scope of protection of the present invention.

[0042] As mentioned above, existing methods for constructing single-stranded nucleic acid circular libraries all require connecting adapters to both ends of the nucleic acid template, which increases the experimental steps and costs, while also resulting in the loss of the original nucleic acid template and reducing the utilization rate of the nucleic acid template.

[0043] Therefore, the present invention aims to provide a novel method for constructing a single-stranded nucleic acid circular library. The inventors have optimized and improved existing single-stranded library construction techniques by combining multiple sequencing primer binding sequences within an adapter molecule or separately including multiple sequencing primer binding sequences within an adapter molecule and an extension primer. This method forms a partially double-stranded adapter element between the adapter molecule and the auxiliary strand, which is then ligated to the end (3' or 5') of the single-stranded nucleic acid template via a ligase, thereby obtaining partially double-stranded nucleic acid fragments. Optionally, the extension primer is then used to perform an extension reaction, and the resulting product is subjected to a single-stranded circularization reaction, thereby completing the present invention.

[0044] In a first aspect, the present invention provides a method for constructing a single-stranded nucleic acid circular library, comprising the following steps:

[0045] 1) Providing a single-stranded nucleic acid template;

[0046] 2) forming a partially double-stranded linker element between the linker molecule and the auxiliary chain, wherein the 5' end portion of the auxiliary chain is reverse complementary to the 5' end portion of the linker molecule, and the 3' end of the auxiliary chain has a random base sequence that is hybridized and complementary to the 3' end portion of the single-stranded nucleic acid template;

[0047] 3) ligating the linker molecule in the linker element to the 3' end of the single-stranded nucleic acid template using a ligase, thereby obtaining a nucleic acid fragment with a partial double strand;

[0048] 4) Performing a single-stranded circularization reaction on the product in step 3) to obtain a single-stranded nucleic acid circular library.

[0049] In a second aspect, the present invention further provides a method for constructing a single-stranded nucleic acid circular library, comprising the following steps:

[0050] 1) Providing a single-stranded nucleic acid template;

[0051] 2) forming a partially double-stranded linker element between the linker molecule and the auxiliary chain, wherein the 3' end portion of the auxiliary chain is reverse complementary to the 3' end portion of the linker molecule, and the 5' end of the auxiliary chain has a random base sequence that is hybridized and complementary to the 5' end portion of the single-stranded nucleic acid template;

[0052] 3) ligating the linker molecule in the linker element to the 5' end of the single-stranded nucleic acid template using a ligase, thereby obtaining a nucleic acid fragment with a partial double strand;

[0053] 4) Performing a single-stranded circularization reaction on the product obtained in step 3) to obtain a single-stranded nucleic acid circular library.

[0054] The methods for constructing the single-stranded nucleic acid circular libraries according to the first and second aspects of the present invention are described in detail below.

[0055] In step 1), a single-stranded nucleic acid template is provided.

[0056] The single-stranded nucleic acid template used in the present invention can be a single-stranded DNA template or a single-stranded RNA template, for example, it can be a DNA fragment of 100 bp to 500 bp obtained by fragment screening after fragmentation of intact genomic DNA, it can be an RNA fragment obtained by fragmentation of extracted total RNA (including mRNA, IncRNA and small RNA, etc.), it can be an extracellular free nucleic acid obtained by extraction, it can be a degraded nucleic acid extracted from various degraded biological samples such as formalin-fixed biological tissue samples, paraffin-embedded biological tissue samples, forensic samples or paleontological fossils, it can also be a DNA fragment after fragmentation of intact genomic DNA or a nucleic acid purified and recovered after sulfite treatment of extracellular free nucleic acid, without limitation thereto. Therefore, in one embodiment, the single-stranded nucleic acid template can be a DNA fragment obtained by fragmentation of intact genomic DNA that has been denatured, an RNA fragment obtained by fragmentation of extracted total RNA, an extracellular free nucleic acid, a degraded nucleic acid extracted from a degraded biological sample, or a nucleic acid treated with sulfite. In some embodiments, the degraded biological samples may include formalin-fixed (FFPE) and paraffin-embedded biological tissue samples, forensic samples, and paleontological fossils. As described in the background art, the nucleic acids extracted from these samples are generally low in quantity, have degradation phenomena, and contain both double-stranded and single-stranded nucleic acid molecules, making it impossible to use conventional library construction methods that can only utilize complete double-stranded nucleic acid molecules. The library construction method of the present invention can utilize single-stranded nucleic acid templates for library construction, thereby minimizing the loss of irregular double-stranded nucleic acid template molecules, thereby maximizing the utilization rate of nucleic acid templates. However, the single-stranded nucleic acid templates that can be used in the present invention are not limited thereto. Any suitable sample can be used in the present invention.

[0057] Furthermore, it is understood that when the nucleic acid template is a double-stranded nucleic acid rather than a single-stranded nucleic acid, it also needs to be denatured. Conventional denaturation methods in the art can be used to denature double-stranded nucleic acids. For example, the DNA sample can be placed at 92°C to 98°C for 3-10 minutes to melt the double-stranded DNA into single-stranded DNA. In addition, in order to prevent the melted single-stranded DNA from renaturing, the sample needs to be cooled immediately after denaturation, for example by placing the sample on ice.

[0058] In step 2), a linker element having a partial double strand is formed between the linker molecule and the auxiliary chain.

[0059] The adapter molecule can be attached to the 5' or 3' end of the single-stranded nucleic acid template, depending on the specific situation. For example, if the nucleic acid template is sufficient and PCR amplification is not required, attaching the adapter molecule to either the 5' or 3' end of the single-stranded nucleic acid template is feasible. However, if the nucleic acid template is insufficient and PCR amplification is required, the adapter molecule should be attached to the 3' end of the single-stranded nucleic acid template to achieve PCR amplification of the single-stranded nucleic acid template.

[0060] In one embodiment, the 5' end portion of the auxiliary chain has a reverse complementary sequence to the 5' end portion of the adapter molecule, and the 3' end of the auxiliary chain has a random base sequence that hybridizes complementary to the 3' end portion of the single-stranded nucleic acid template. In this case, the adapter molecule is attached to the 3' end of the single-stranded nucleic acid template, and the subsequent determination of whether to perform PCR amplification can be based on the amount of template.

[0061] In another embodiment, the 3' end portion of the auxiliary chain has a reverse complementary sequence to the 3' end portion of the adapter molecule, and the 5' end of the auxiliary chain has a random base sequence that hybridizes complementary to the 5' end portion of the single-stranded nucleic acid template. In this case, the adapter molecule is attached to the 5' end of the single-stranded nucleic acid template. Because this situation cannot be used to amplify the single-stranded nucleic acid template through PCR, it is only suitable when there is sufficient template.

[0062] In one embodiment, the 5' end of the adapter molecule is phosphorylated and the 3' end of the adapter molecule is modified and blocked. The purpose of phosphorylation modification of the 5' end of the adapter molecule is to enable the adapter molecule to react with the single-stranded nucleic acid template. In addition, when the adapter molecule is connected to the 3' end of the single-stranded nucleic acid template, the 3' end of the adapter molecule needs to be modified and blocked, the purpose of which is to avoid self-ligation and non-specific amplification between the adapters, greatly improve the utilization rate of the template in the library construction, and improve the specificity of the library. In one embodiment, the modification for blocking includes phosphorylation modification, C3-spacer modification, amino modification and C6 Spacer modification. In one embodiment, the 3' end of the adapter molecule has a reversible blocking group. In this article, the so-called "reversible blocking group" refers to a blocking group that can be reversibly removed from the adapter molecule later. In the case of subsequent PCR amplification, since the original nucleic acid template content becomes relatively low, the final cyclization reaction can be carried out directly without removing the blocking group on the adapter molecule; and in the case of subsequent PCR amplification without PCR amplification, it is preferred to remove the blocking group on the adapter molecule before carrying out the final cyclization reaction. Furthermore, when the adaptor molecule is linked to the 5' end of the single-stranded nucleic acid template, the 3' end of the adaptor molecule is not blocked in order to allow for ligation to the single-stranded nucleic acid template.

[0063] The library construction method of the present invention involves the use of auxiliary chains. In order to achieve the connection between the auxiliary nucleic acid template and the adapter molecule, one end of the auxiliary chain needs to have a random base sequence that hybridizes complementary to the partial sequence of the connected end of the nucleic acid template, while the partial sequence of the other end needs to be reverse complementary to the partial sequence of the connected end of the adapter molecule. More specifically, in the case where the adapter molecule is intended to be connected to the 3' end of the nucleic acid template, the partial sequence at the 5' end of the auxiliary chain is reverse complementary to the partial sequence at the 5' end of the adapter molecule, and the 3' end of the auxiliary chain has a random base sequence that hybridizes complementary to the 3' end of the nucleic acid template; in the case where the adapter molecule is intended to be connected to the 5' end of the nucleic acid template, the partial sequence at the 3' end of the auxiliary chain is reverse complementary to the partial sequence at the 3' end of the adapter molecule, and the 5' end of the auxiliary chain has a random base sequence that hybridizes complementary to the 5' end of the nucleic acid template. Through this structure, a linker element with a partial double strand is first formed between the adapter molecule and the auxiliary chain, and then a ligase can be used to connect the single-stranded nucleic acid template and the linker element together, thereby forming a nucleic acid fragment with a partial double strand.

[0064] The 3' end of the auxiliary chain can also be modified and blocked as needed. In one embodiment, the 3' end of the auxiliary chain is modified and blocked, and the modifications include phosphorylation modification, C3-spacer modification, amino modification, and C6 spacer modification to prevent self-ligation between auxiliary chains and nonspecific extension using the auxiliary chain as a primer.

[0065] In one embodiment, the sequence portion of the auxiliary chain that is reverse complementary to the linker molecule can have 15-30 nucleotides, such as 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides, preferably 20 nucleotides.

[0066] In another embodiment, the complementary random base sequence of the auxiliary chain hybridizing to the target DNA template may have 6-12 nucleotides, such as 6, 7, 8, 9, 10, 11 or 12 nucleotides, preferably 12 nucleotides.

[0067] In one embodiment, the adapter molecule may comprise at least one sequencing primer binding sequence or a combination of multiple sequencing primer binding sequences.

[0068] As mentioned in the background section, in the prior art, two sequencing adapters need to be connected to the nucleic acid fragment separately in order to achieve sequencing of the nucleic acid fragment. However, in the method of the present invention, the two sequencing primer binding sequences used in the sequencing platform can be first connected to form an adapter molecule, and then the adapter molecule can be connected to the single-stranded nucleic acid template. In this case, the adapter molecule comprises a combination of two sequencing primer binding sequences. In addition, in the method of the present invention, if the adapter molecule only comprises one sequencing primer binding sequence, an extension reaction is required to connect the other sequencing primer binding sequence to the one sequencing primer binding sequence and the single-stranded nucleic acid template, as described below.

[0069] In addition, in one embodiment, the adapter molecule may further comprise a tag sequence, comprising a sample tag for distinguishing the source of the sample or a unique molecular tag for distinguishing nucleic acid molecules. In one embodiment, the tag sequence may be located between multiple sequencing primer binding sequences in the adapter molecule.

[0070] In step 3), the linker molecule in the linker element is ligated to the end of the single-stranded nucleic acid template by a ligase, thereby obtaining a nucleic acid fragment with a partial double strand.

[0071] Those skilled in the art can select specific ligases according to the type of single-stranded nucleic acid template. In one embodiment, the ligase can be a DNA ligase, including but not limited to T4 DNA ligase, T3 DNA ligase, Taq DNA ligase, etc., preferably T4 DNA ligase. In one embodiment, the ligase can be an RNA ligase, including but not limited to T4 RNA ligase 2.

[0072] In one embodiment, when the single-stranded nucleic acid template is a single-stranded RNA template, the library construction method further comprises a step 3') between steps 3) and 4): reverse transcribing the single-stranded RNA template using an extension primer complementary to the 3' end sequence of the adapter molecule and a reverse transcriptase.

[0073] First, it is important to note that step 3') only applies to embodiments in which an adapter molecule is attached to the 3' end of a single-stranded nucleic acid template. Second, whether the method of the present invention includes step 3') depends on the type of single-stranded nucleic acid template employed. Specifically, when employing a single-stranded DNA template, step 3') is unnecessary. However, when employing a single-stranded RNA template, the RNA must be reverse transcribed into DNA before proceeding to the next step and subsequent steps. Conventional reverse transcription techniques in the art can be used to reverse transcribe RNA into DNA, and these techniques will not be further described herein.

[0074] In one embodiment, the library construction method of the present invention further comprises, before step 4), step 3″): performing a linear extension reaction on the partially double-stranded nucleic acid fragment obtained in step 3) using an extension primer, wherein the extension primer is reverse complementary to the 3' end sequence of the adapter molecule. It should be understood that when the adapter molecule is connected to the 3' end of the single-stranded nucleic acid template, this step is optional. In other words, the library construction method of the present invention may include this step, which is particularly suitable for situations where the nucleic acid sample amount is insufficient. The library construction method of the present invention may also exclude this step, which is particularly suitable for situations where the nucleic acid sample amount is sufficient. In situations where the nucleic acid sample amount is sufficient, the adapter molecule may also be connected to the 5' end of the single-stranded nucleic acid template, in which case the method of the present invention does not include the extension reaction step.

[0075] To initiate a linear extension reaction, the 5' end of the extension primer must be reverse complementary to the 3' end of the adapter to ensure smooth initiation of the extension reaction. Additionally, to prevent unwanted self-ligation between extension primers, the 5' end of the extension primer can be phosphorylated.

[0076] Furthermore, it is understood that, when performing an extension reaction, the auxiliary chain can be used directly as the extension primer. Therefore, in one embodiment, the 3' end of the auxiliary chain has a reversible blocking group. Thus, when performing an extension reaction, the reversible blocking group at the 3' end of the auxiliary chain can be removed first, and then the extension reaction can be performed using the auxiliary chain as the extension primer, without the need to introduce an additional extension primer.

[0077] As a further embodiment, the adapter molecule may only contain a sequencing primer binding sequence, and the extension primer may contain a sequence that is reverse complementary to the sequencing primer binding sequence at the 3' end, and another sequencing primer binding sequence at the 5' end. It is understandable that in this case, the linear extension reaction of step 3") is necessary, the purpose of which is to completely introduce the sequencing primer binding sequence through the extension reaction. Specifically, in the method of the present invention, the first sequencing primer binding sequence for the sequencing platform can be first connected to the nucleic acid template, and then an extension primer containing a second sequencing primer binding sequence and a sequence that is reverse complementary to the 3' end of the first sequencing primer binding sequence can be used to connect the second sequencing primer binding sequence to the first sequencing primer binding sequence and the single-stranded nucleic acid template through PCR amplification; a partial sequence of the first sequencing primer binding sequence for the sequencing platform can be first connected to the nucleic acid template, and then a portion of the sequence of the first sequencing primer binding sequence and the remaining sequence of the first sequencing primer binding sequence can be used. The second sequencing primer binding sequence and the remaining sequence of the first sequencing primer binding sequence are ligated together with the partial sequence of the first sequencing primer binding sequence and the single-stranded nucleic acid template by PCR amplification using an extension primer containing a sequence that is reverse complementary to the 3' end of the partial sequence of the second sequencing primer binding sequence. Alternatively, the first sequencing primer binding sequence used in the sequencing platform and the partial sequence of the second sequencing primer binding sequence can be first ligated to the nucleic acid template, and then an extension primer containing the remaining sequence of the second sequencing primer binding sequence and a sequence that is reverse complementary to the 3' end of the partial sequence of the second sequencing primer binding sequence can be used to ligate together with the first sequencing primer binding sequence, the partial sequence of the second sequencing primer binding sequence and the single-stranded nucleic acid template by PCR amplification.

[0078] In some embodiments, the extension primer may further comprise a tag sequence, comprising a sample tag for distinguishing the source of the sample or a unique molecular tag for distinguishing nucleic acid molecules. The tag sequence can be linked to the adapter molecule and the single-stranded nucleic acid template through an extension reaction.

[0079] In addition, the linear extension reaction can be carried out according to conventional methods known in the art, and the number of cycles can be adjusted according to the amount of nucleic acid input, and the general number of cycles is 5 to 30.

[0080] In step 4), the product in step 3) or 3') is subjected to a single-stranded circularization reaction to obtain a single-stranded nucleic acid circular library. The single-stranded circularization reaction can be performed using, for example, a single-stranded circular ligase.

[0081] In step 4), the DNA fragment with local double strands obtained in step 3) or 3') or the linear extension product obtained in step 3") can be denatured first and then subjected to a single-stranded cyclization reaction; or the denaturation treatment and the single-stranded cyclization reaction can be performed simultaneously. The double-stranded DNA formed can be denatured by conventional denaturation methods in the art. For example, the DNA fragment with local double strands or the extension product can be placed at 92°C to 98°C for 3-10 minutes to melt the double-stranded DNA into single-stranded DNA. In addition, in order to prevent the single-stranded DNA obtained by melting from renaturing, the sample needs to be cooled immediately after the denaturation is completed, for example, by placing the sample on ice. Through this denaturation treatment, a single-stranded DNA in which the single-stranded DNA template is connected to the adapter molecule can be obtained.

[0082] Furthermore, before proceeding to the next step, the obtained product can be purified to screen out the target product and prevent the influence and interference of non-target products on subsequent methods. Therefore, in one embodiment, the library construction method further includes purifying the product obtained in at least one of steps 3), 3'), 3"), and 4), for example, by magnetic bead purification.

[0083] It is understood that after step 4), some uncircularized single-stranded nucleic acid templates may remain. To ensure more reliable subsequent sequencing, these remaining single-stranded nucleic acid templates can be processed. Therefore, in one embodiment, the library construction method further comprises: digesting the uncircularized single-stranded nucleic acid using a linear digestion enzyme after the single-stranded nucleic acid is circularized in step 4).

[0084] The single-stranded nucleic acid circular library obtained by the above-mentioned library construction method of the present invention can be used for sequencing.

[0085] It should be noted that, at least for the methods of the first or second aspects of the present invention, although the steps are labeled with numbers 1, 2, etc., it should be understood that the numbers are merely for the purpose of distinguishing the steps and are not intended to indicate a chronological order. Specifically, the steps of the present invention can be performed in any order, such as sequentially, simultaneously, or in reverse order, as long as the method is ultimately implemented. Adjusting the order of the steps of the present invention is within the capabilities of those skilled in the art.

[0086] Therefore, in a third aspect, the present invention provides a method for sequencing single-stranded circular DNA, the method comprising:

[0087] a) performing the method for constructing a single-stranded nucleic acid circular library according to the first or second aspect of the present invention to obtain a single-stranded nucleic acid circular library;

[0088] b) sequencing the single-stranded nucleic acid circular library product obtained in step a) to obtain sequencing data.

[0089] For the specific details of the method for constructing the single-stranded nucleic acid circular library described in the first aspect or the second aspect of the present invention in step a), reference can be made to the above description of these two aspects and will not be repeated here.

[0090] In a fourth aspect, the present invention provides a kit for constructing a single-stranded nucleic acid circular library, the kit comprising:

[0091] (1) Linker molecules;

[0092] (2) an auxiliary chain, wherein one end of the auxiliary chain has a sequence that is reverse complementary to a portion of the sequence at the connecting end of the linker molecule, and the other end of the auxiliary chain has a random base sequence that is complementary to the hybridization end of the single-stranded nucleic acid template;

[0093] (3) Ligase.

[0094] In one embodiment, the adapter molecule may comprise at least one sequencing primer binding sequence or a combination of multiple sequencing primer binding sequences.

[0095] In one embodiment, the length of the random base sequence contained in the auxiliary chain can be 6-12 nucleotides, preferably 12 nucleotides.

[0096] In one embodiment, the ligase may be a DNA ligase, including but not limited to T4 DNA ligase, T3 DNA ligase, Taq DNA ligase, etc., preferably T4 DNA ligase. In one embodiment, the ligase may be an RNA ligase, including but not limited to T4 RNA ligase 2.

[0097] In an optional embodiment, the kit may further comprise an extension primer comprising a sequence that is reverse complementary to the adapter molecule. In a further optional embodiment, the extension primer further comprises a sequencing primer binding sequence, wherein the sequence that is reverse complementary to the adapter molecule is located at the 3' end of the extension primer, and the sequencing primer binding sequence is located at the 5' end of the extension primer.

[0098] In one embodiment, the 5' end of the adapter molecule and the 5' end of the extension primer are phosphorylated, and the 3' end of the auxiliary chain is modified and blocked. In an optional embodiment, the 3' end of the adapter molecule is also modified and blocked. In one embodiment, the modifications used for blocking include phosphorylation modification, C3-spacer modification, amino modification, and C6 spacer modification.

[0099] In one embodiment, the adapter molecule and / or the extension primer further comprises a tag sequence, wherein the tag sequence comprises a sample tag for distinguishing the source of a sample or a unique molecular tag for distinguishing a nucleic acid molecule.

[0100] In a further embodiment, the tag sequence is located between multiple sequencing primer binding sequences of the adapter molecule.

[0101] It is understood that, in addition to the above components, the kit may also contain other reagents, such as polymers, buffers, nucleotide mixtures, and the like.

[0102] The linker molecules, auxiliary chains, ligases and extension primers in the kit of this aspect of the invention are the same as or similar to the linker molecules, auxiliary chains, ligases and extension primers in the first, second and third aspects of the invention, and therefore will not be described further.

[0103] The present invention will be further described below by the following examples. Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the materials and reagents used in the following examples are all commercially available.

[0104] Example

[0105] Example 1: Construction and sequencing of human peripheral blood cell-free DNA library

[0106] 1. Experimental Materials

[0107] Free DNA extracted from human peripheral blood plasma

[0108] 2. Experimental steps

[0109] 1. Transfer 5 ng of free DNA from human peripheral blood plasma to a 200 μL PCR tube and add enzyme-free water to a total volume of 45 μL. Denature the DNA in a PCR instrument (95°C for 3 minutes) and immediately place on ice.

[0110] 2. Prepare the adapter-auxiliary strand mixture: anneal and hybridize 20 μM of adapter molecules (the combined adapter used to connect the 5' and 3' sequencing adapters on the DNBSEQ sequencing platform) and 20 μM of auxiliary strand at a 1:1 ratio to form a partially double-stranded adapter element. The reaction conditions are: 95°C for 3 minutes, followed by cooling to 4°C, with the PCR instrument set to a cooling rate of 0.1°C / second.

[0111] Sequence of the linker molecule:

[0112] 5'Pho-AAGTCGGATCGTAGCCATGTCGTTCTGTGAGCCAAGGAGTTGATCGGACCTATTGTCTTCCTAAGACCGCTTGGCCTCCGACTT-3'Pho

[0113] Sequence of auxiliary chain:

[0114] 5'-ACATGGCTACGATCCGACTTNNNNNNNNNNNN-3'C3-spacer

[0115] 3. Use T4 DNA ligase and ligation buffer from the MGI DNA library preparation kit (1000005254, MGI) to ligate the adapter and DNA template. Prepare the reaction solution on ice according to Table 1 below.

[0116] Table 1. Adapter ligation reaction solution

[0117] Component volume T4 DNA ligase 1.6 μL Ligation buffer 23.4 μL Adapter element 10 μL

[0118] 4. Add the prepared adapter ligation reaction solution (35 μL) to the PCR tube containing the denatured DNA, mix thoroughly using a shaker, and then centrifuge briefly to centrifuge the liquid to the bottom of the tube.

[0119] 5. Place the centrifuged PCR tube on a PCR instrument and react at 30°C for 30 minutes to perform the ligation reaction.

[0120] 6. After the ligation reaction is complete, transfer the reaction product to a 1.5 mL centrifuge tube. Then, add 96 μL of purified magnetic beads to the 1.5 mL centrifuge tube. Pipette 10 times to mix thoroughly, and incubate at room temperature for 5 minutes.

[0121] 7. After another instant centrifugation, place the 1.5 mL centrifuge tube on a magnetic stand and let it stand for 5 minutes until the liquid becomes clear. Then use a pipette to aspirate the supernatant and discard it.

[0122] 8. Add 200 μL of 75% ethanol to the 1.5 mL centrifuge tube and let it sit for 30 seconds to wash the magnetic beads. Repeat the washing steps once.

[0123] 9. Try to dry the liquid in the centrifuge tube. If a small amount of liquid remains on the wall of the tube, the 1.5 mL centrifuge tube can be centrifuged instantly. After separation on the magnetic stand, use a small-scale pipette to dry the liquid at the bottom of the tube.

[0124] 10. Keep the 1.5 mL centrifuge tube fixed on the magnetic rack, open the centrifuge tube cap, and dry it at room temperature until the surface of the magnetic beads is non-reflective and free of cracks.

[0125] 11. Remove the 1.5 mL centrifuge tube from the magnetic stand and add 23 μL of molecular-grade water to elute the DNA. Gently pipette 10 times to mix thoroughly, then incubate at room temperature for 5 minutes.

[0126] 12. Centrifuge briefly, place the 1.5 mL centrifuge tube on a magnetic stand, and let it stand for 5 minutes until the liquid becomes clear. Then transfer 22 μL of the supernatant to a new 200 μL PCR tube.

[0127] 13. Use 20 μM extension primer-L and the PCR enzyme mixture from the MGI enzyme digestion DNA library preparation kit (1000005254, MGI) to prepare the extension reaction solution according to Table 2 below.

[0128] Extension Primer-L: 5'Pho-AAGTCGGAGGCCAAGCG-3'

[0129] Table 2. Extension reaction solution

[0130] Component volume PCR enzyme mix 25μL PCR primer -L 3μL

[0131] 14. Add the prepared extension reaction solution (28 μL) to the PCR tube containing the purified ligation product, mix thoroughly using a vortex mixer, and then centrifuge briefly to centrifuge the liquid to the bottom of the tube.

[0132] 15. Place the centrifuged PCR tube on a PCR instrument and perform the reaction according to Table 3 below.

[0133] Table 3. Extension reaction conditions

[0134]

[0135] 16. After the extension reaction is completed, transfer the reaction product to a 1.5 mL centrifuge tube. Then add 60 μL of purified magnetic beads to the centrifuge tube. Pipette 10 times to mix thoroughly and incubate at room temperature for 5 minutes.

[0136] 17. After another instant centrifugation, place the 1.5 mL centrifuge tube on a magnetic stand and let it stand for 5 minutes until the liquid becomes clear. Use a pipette to aspirate the supernatant and discard it.

[0137] 18. Add 200 μL of 75% ethanol to the 1.5 mL centrifuge tube and let it sit for 30 seconds to wash the magnetic beads. Repeat the washing step once.

[0138] 19. Try to dry the liquid in the centrifuge tube. If a small amount of liquid remains on the tube wall, the 1.5 mL centrifuge tube can be centrifuged instantly. After separation on the magnetic stand, use a small-scale pipette to dry the liquid at the bottom of the tube.

[0139] 20. Keep the 1.5 mL centrifuge tube fixed on the magnetic rack, open the centrifuge tube cap, and dry it at room temperature until the surface of the magnetic beads is non-reflective and free of cracks.

[0140] 21. Remove the 1.5 mL centrifuge tube from the magnetic stand and add 18 μL of molecular-grade water to elute the DNA. Gently pipette 10 times to mix thoroughly, then incubate at room temperature for 5 minutes.

[0141] 22. After brief centrifugation, place the 1.5 mL centrifuge tube on a magnetic stand and let it stand for 5 minutes until the liquid becomes clear. Then transfer 16.5 μL of the supernatant to a new 200 μL PCR tube.

[0142] 23. Place the PCR tube on a PCR instrument for denaturation (95°C for 3 minutes), and then immediately place it on ice. Use a fluorescence quantitative kit ( ssDNA Assay Kit) and 1 μL of PCR product was taken for quantification according to the operating instructions of the quantification kit.

[0143] 24. Circularize the obtained single-stranded DNA using the CircLigase kit (CL4111K, epicentre) to form a single-stranded DNA circular library. Prepare the single-stranded circularization reaction solution according to Table 4 below.

[0144] Table 4. Single-chain cyclization reaction solution

[0145] Component Volume 10X Reaction Buffer 2 μL CircLigase 1 μL 50 mM MnCl 2 1 μL 1 mM ATP 0.5 μL

[0146] 25. Add the prepared single-stranded circularization reaction solution to the PCR tube containing the denatured PCR product, mix thoroughly using a vortex mixer, and then centrifuge briefly to centrifuge the liquid to the bottom of the tube.

[0147] 26. Place the centrifuged PCR tube on a PCR instrument and perform the reaction according to the conditions in Table 5 below.

[0148] Table 5. Single-chain circularization reaction conditions

[0149] Temperature Time 105℃ Hot lid open 60℃ 1 hour 85℃ 10 minutes 4℃∞

[0150] 27. After the reaction is completed, centrifuge the PCR tube briefly and place it on ice before proceeding to the next step.

[0151] 28. Use the circularization kit from MGI (1000005260, MGI) and prepare the enzyme digestion reaction solution on ice in advance according to the recipe in Table 6 below.

[0152] Table 6. Enzyme digestion reaction solution

[0153] Components Single reaction volume Digestion buffer 1.4 μL Digestion enzyme 2.6 μL Total volume 4 μL

[0154] 29. Use a pipette to draw 4 μL of the prepared enzyme digestion reaction solution into the PCR tube. Vortex 3 times for 3 seconds each time. Centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0155] 30. Place the PCR tube on a PCR instrument and perform the reaction according to the conditions in Table 7 below.

[0156] Table 7. Enzymatic digestion reaction conditions

[0157] Temperature Time 75℃ Hot lid open 37℃ 30 minutes 4℃∞

[0158] 31. After the digestion reaction is completed, collect the reaction solution to the bottom of the tube by instant centrifugation.

[0159] 32. Immediately add 6 μL of digestion stop buffer (1000005260, MGI) to the PCR tube, vortex 3 times for 3 seconds each time, centrifuge briefly to collect the reaction solution at the bottom of the tube, and transfer all the reaction solution to a new 1.5 mL centrifuge tube.

[0160] 33. Then add 72 μL of purified magnetic beads to the 1.5 mL centrifuge tube, pipette 10 times to mix thoroughly, and incubate at room temperature for 5 minutes.

[0161] After brief centrifugation, place the 1.5 mL centrifuge tube on a magnetic stand and let it stand for 5 minutes until the liquid becomes clear. Pipette the supernatant and discard it.

[0162] 35. Add 200 μL of 75% ethanol to the 1.5 mL centrifuge tube and let it sit for 30 seconds to wash the magnetic beads. Repeat the washing step once.

[0163] 36. Try to drain the liquid in the centrifuge tube. If there is a small amount of liquid remaining on the wall of the tube, you can centrifuge the 1.5mL centrifuge tube briefly. After separation on the magnetic stand, use a small-scale pipette to drain the liquid at the bottom of the tube.

[0164] 37. Keep the 1.5 mL centrifuge tube fixed on the magnetic rack, open the centrifuge tube cap, and dry it at room temperature until the surface of the magnetic beads is non-reflective and free of cracks.

[0165] Remove the 1.5 mL centrifuge tube from the magnetic stand and add 21 μL of molecular-grade water to elute the DNA. Pipette gently 10 times to mix thoroughly, then incubate at room temperature for 5 minutes.

[0166] 39. After the brief centrifugation, place the 1.5 mL centrifuge tube on a magnetic stand and let it stand for 5 minutes until the liquid becomes clear. Then transfer 20 μL of the supernatant to a new 1.5 mL centrifuge tube.

[0167] 40. Use the fluorescence quantitative kit ( ssDNA Assay Kit) and quantify the purified products after enzyme digestion according to the operating instructions of the quantification kit.

[0168] 41. Use MGISEQ-2000 High-Throughput Sequencing Reagent Set (PE100) (MGI, Cat. No. 1000012536) to prepare DNA nanoballs (DNBs) according to the instructions.

[0169] 42. Use the fluorescence quantitative kit ( ssDNA Assay Kit) and quantify DNB according to the operating instructions of the quantification kit.

[0170] 3. Experimental Results

[0171] The experimental results are shown in Table 8. As can be seen from this table, the product concentration after linear extension and purification was 6.5 ng / μL. After single-stranded circularization of the extension product and linear DNA digestion, the final single-stranded circular DNA library concentration was 1.4 ng / μL. 8 ng of the single-stranded circular library was used to prepare DNA nanoballs (DNBs) unique to the DNBSEQ platform, resulting in a DNB concentration of 18.4 ng / μL, which met the standard and confirmed that the single-stranded circular library was qualified.

[0172] Table 8. Experimental results

[0173]

[0174] Example 2: Construction and sequencing of a human peripheral blood free DNA methylation library

[0175] 1. Experimental Materials

[0176] Free DNA extracted from human peripheral blood plasma

[0177] 2. Experimental steps

[0178] 1. Take 5 ng of human peripheral blood plasma free DNA (cfDNA) and transfer it to a 200 μL PCR tube, with a total volume of 20 μL.

[0179] 2. cfDNA was bisulfite treated and purified using the EZ DNA Methylation-Gold Kit (Zymo Research, Cat. No. D5005 / D5006).

[0180] 3. Prepare the CT Conversion Reagent by pipetting 900 μL of nanofiltered water, 300 μL of M-Dilution Buffer, and 50 μL of M-Lyzing Buffer into a tube of CT Conversion Reagent powder (centrifuge briefly before opening the tube). Vortex frequently at room temperature for 10 minutes to complete the preparation of the CT Conversion Reagent. Minimize exposure to light and prepare the CT Conversion Reagent immediately. The CT Conversion Reagent can be stored at room temperature for up to 1 day, at 4°C for up to 1 week, and at -20°C for up to 1 month. Non-freshly prepared CT Conversion Reagent should be prewarmed to 37°C and vortexed frequently for 10 minutes at room temperature before use.

[0181] 4. Before opening the bottle for the first time, add the correct volume of anhydrous ethanol as indicated on the bottle label and mix thoroughly before use. At room temperature, add the ingredients listed in Table 9 to a new 200 μL PCR tube.

[0182] Table 9. Reagents and samples for bisulfite treatment

[0183] Component Volume CT Conversion Reagent 130μL cfDNA 20μL Total Volume 150μL

[0184] 5. Place the 200 μL PCR tube in a PCR instrument and perform the reaction according to the bisulfite treatment conditions in Table 10.

[0185] Table 10. Bisulfite treatment conditions

[0186] Temperature Time: Heated lid open 98℃ 10 minutes 64℃ 2.5 hours 4℃ ∞

[0187] 6. Transfer the reaction product to a new 1.5 mL centrifuge tube, add 600 μL of M-binding buffer, vortex 6 times for 3 seconds each time, and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0188] 7. Place the Zymo-Spin IC column in a 2 mL collection tube, transfer the mixture to the Zymo-Spin IC column, centrifuge at 13,000 rpm for 30 seconds, discard the waste liquid, and place the Zymo-Spin IC column back into the collection tube.

[0189] 8. Add 100 μL of M-wash buffer to the Zymo-Spin IC column and centrifuge at 13,000 rpm for 30 seconds.

[0190] 9. Add 200 μL of M-desulfonation buffer to the Zymo-Spin IC column. Quickly cap the tube and incubate at room temperature for 15 to 20 minutes. Centrifuge at 13,000 rpm for 30 seconds, discard the waste liquid, and return the Zymo-Spin IC column to the collection tube.

[0191] 10. Add 200 μL of M-wash buffer to the Zymo-Spin IC column, centrifuge at 13,000 rpm for 30 seconds, discard the waste liquid, and return the Zymo-Spin IC column to the collection tube.

[0192] 11. Add 200 μL of M-Wash Buffer to the Zymo-Spin IC column and centrifuge at 13,000 rpm for 30 seconds. Discard the waste liquid and return the Zymo-Spin IC column to the collection tube. Centrifuge at 13,000 rpm for 30 seconds without rotation. Discard the collection tube and use a pipette to remove as much liquid as possible from the outer wall of the Zymo-Spin IC column. Place the column in a new 1.5 mL centrifuge tube.

[0193] 12. Open the Zymo-Spin IC column cap and dry it at room temperature for 2 minutes. Then place the Zymo-Spin IC column in another new 1.5 mL centrifuge tube.

[0194] 13. Slowly add 10 μL of M-elution buffer to the center of the Zymo-Spin IC column filter, let it stand for 1 minute, and centrifuge at 13,000 rpm for 30 seconds. The purified product after bisulfite treatment is collected in a 1.5 mL centrifuge tube.

[0195] 14. Place all the bisulfite-treated and purified products in a new 200 μL PCR tube and add molecular-grade water to make up the total volume to 20 μL.

[0196] 15. Subsequently, all experimental steps in Example 1 were carried out on the 20 μL sample thus obtained.

[0197] 16. Use MGISEQ-2000 sequencer for sequencing, use MGISEQ-2000 high-throughput sequencing reagent set (PE100) (MGI, product number 1000012536), and follow the instructions for DNB production and sequencing. ssDNA Assay Kit fluorescence quantification kit was used to quantify DNB according to the operating instructions of the quantification kit.

[0198] 3. Experimental Results

[0199] a. Database construction results

[0200] The specific results are shown in Table 11. As can be seen from this table, after sulfite conversion of cfDNA and linear extension followed by purification, the resulting product concentration was 7.8 ng / μL. After single-stranded circularization of the extension product and linear DNA digestion, the final single-stranded circular DNA library concentration was 2.3 ng / μL. 8 ng of the single-stranded circular library was used to prepare DNA nanoballs (DNBs), a unique feature of the DNBSEQ platform. The resulting DNB concentration was 18.1 ng / μL, meeting the standard and confirming that the single-stranded circular library was qualified.

[0201] Table 11. Library construction results

[0202]

[0203] b. Sequencing results

[0204] After the DNB prepared in Example 2 was sequenced, the obtained data was analyzed using a conventional whole-genome methylation sequencing analysis process, including filtering of low-quality data, alignment with the reference genome, and statistics on coverage, conversion rate, and number of CG sites to obtain the corresponding analysis results (Table 12).

[0205] Table 12. Sequencing results

[0206]

[0207]

[0208] Example 3: Construction and sequencing of human peripheral blood cell-free DNA library

[0209] 1. Experimental Materials

[0210] Free DNA extracted from human peripheral blood plasma

[0211] 2. Experimental steps

[0212] 1. Transfer 5 ng of free DNA from human peripheral blood plasma to a 200 μL PCR tube and add enzyme-free water to a total volume of 45 μL. Denature the DNA in a PCR instrument (95°C for 3 minutes) and immediately place on ice.

[0213] 2. Prepare the adapter-auxiliary strand mixture: anneal and hybridize 20 μM of adapter molecules (the combined adapter used to connect the 5' and 3' sequencing adapters on the DNBSEQ sequencing platform) and 20 μM of auxiliary strand at a 1:1 ratio to form a partially double-stranded adapter element. The reaction conditions are: 95°C for 3 minutes, followed by cooling to 4°C, with the PCR instrument set to a cooling rate of 0.1°C / second.

[0214] Sequence of the linker molecule:

[0215] 5'Pho-AAGTCGGATCGTAGCCATGTCGTTCTGTGAGCCAAGGAG-3'Pho

[0216] Sequence of auxiliary chain:

[0217] 5'-ACATGGCTACGATCCGACTTNNNNNNNNNNNN-3'C3-spacer

[0218] 3. Use T4 DNA ligase and ligation buffer from the MGI DNA library preparation kit (1000005254, MGI) to ligate the adapter and DNA template. Prepare the reaction solution on ice according to Table 13 below.

[0219] Table 13. Adapter ligation reaction mixture

[0220] Component volume T4 DNA ligase 1.6 μL Ligation buffer 23.4 μL Adapter element 10 μL

[0221] 4. Add the prepared adapter ligation reaction solution (35 μL) to the PCR tube containing the denatured DNA, mix thoroughly using a shaker, and then centrifuge briefly to centrifuge the liquid to the bottom of the tube.

[0222] 5. Place the centrifuged PCR tube on a PCR instrument and react at 30°C for 30 minutes to perform the ligation reaction.

[0223] 6. After the ligation reaction is complete, transfer the reaction product to a 1.5 mL centrifuge tube. Then, add 96 μL of purified magnetic beads to the 1.5 mL centrifuge tube. Pipette 10 times to mix thoroughly, and incubate at room temperature for 5 minutes.

[0224] 7. After another instant centrifugation, place the 1.5 mL centrifuge tube on a magnetic stand and let it stand for 5 minutes until the liquid becomes clear. Then use a pipette to aspirate the supernatant and discard it.

[0225] 8. Add 200 μL of 75% ethanol to the 1.5 mL centrifuge tube and let it sit for 30 seconds to wash the magnetic beads. Repeat the washing steps once.

[0226] 9. Try to dry the liquid in the centrifuge tube. If a small amount of liquid remains on the wall of the tube, the 1.5 mL centrifuge tube can be centrifuged instantly. After separation on the magnetic stand, use a small-scale pipette to dry the liquid at the bottom of the tube.

[0227] 10. Keep the 1.5 mL centrifuge tube fixed on the magnetic rack, open the centrifuge tube cap, and dry it at room temperature until the surface of the magnetic beads is non-reflective and free of cracks.

[0228] 11. Remove the 1.5 mL centrifuge tube from the magnetic stand and add 23 μL of molecular-grade water to elute the DNA. Gently pipette 10 times to mix thoroughly, then incubate at room temperature for 5 minutes.

[0229] 12. Centrifuge briefly, place the 1.5 mL centrifuge tube on a magnetic stand, and let it stand for 5 minutes until the liquid becomes clear. Then transfer 22 μL of the supernatant to a new 200 μL PCR tube.

[0230] 13. Use 20 μM extension primer-L and the PCR enzyme mixture from the MGI enzyme digestion DNA library preparation kit (1000005254, MGI) to prepare the extension reaction solution according to Table 14 below.

[0231] Extension Primer-L: 5′-AAGTCGGAGGCCAAGCGGTCTTAGGAAGACAATAGGTCCGATCAACTCCTTGGCTCACAGAACGACATG-3′

[0232] Table 14. Extension reaction solution

[0233] Component volume PCR enzyme mix 25μL PCR primer -L 3μL

[0234] 14. Add the prepared extension reaction solution (28 μL) to the PCR tube containing the purified ligation product, mix thoroughly using a vortex mixer, and then centrifuge briefly to centrifuge the liquid to the bottom of the tube.

[0235] 15. Place the centrifuged PCR tube on a PCR instrument and perform the reaction according to Table 15 below.

[0236] Table 15. Extension reaction conditions

[0237]

[0238]

[0239] 16. After the extension reaction is completed, transfer the reaction product to a 1.5 mL centrifuge tube. Then add 60 μL of purified magnetic beads to the centrifuge tube. Pipette 10 times to mix thoroughly and incubate at room temperature for 5 minutes.

[0240] 17. After another instant centrifugation, place the 1.5 mL centrifuge tube on a magnetic stand and let it stand for 5 minutes until the liquid becomes clear. Use a pipette to aspirate the supernatant and discard it.

[0241] 18. Add 200 μL of 75% ethanol to the 1.5 mL centrifuge tube and let it sit for 30 seconds to wash the magnetic beads. Repeat the washing step once.

[0242] 19. Try to dry the liquid in the centrifuge tube. If a small amount of liquid remains on the tube wall, the 1.5 mL centrifuge tube can be centrifuged instantly. After separation on the magnetic stand, use a small-scale pipette to dry the liquid at the bottom of the tube.

[0243] 20. Keep the 1.5 mL centrifuge tube fixed on the magnetic rack, open the centrifuge tube cap, and dry it at room temperature until the surface of the magnetic beads is non-reflective and free of cracks.

[0244] 21. Remove the 1.5 mL centrifuge tube from the magnetic stand and add 18 μL of molecular-grade water to elute the DNA. Gently pipette 10 times to mix thoroughly, then incubate at room temperature for 5 minutes.

[0245] 22. After brief centrifugation, place the 1.5 mL centrifuge tube on a magnetic stand and let it stand for 5 minutes until the liquid becomes clear. Then transfer 16.5 μL of the supernatant to a new 200 μL PCR tube.

[0246] 23. Place the PCR tube on a PCR instrument for denaturation (95°C for 3 minutes), and then immediately place it on ice. Use a fluorescence quantitative kit ( ssDNA Assay Kit) and 1 μL of PCR product was taken for quantification according to the operating instructions of the quantification kit.

[0247] 24. Circularize the obtained single-stranded DNA using the CircLigase kit (CL4111K, epicentre) to form a single-stranded DNA circular library. Prepare the single-stranded circularization reaction solution according to Table 16 below.

[0248] Table 16. Single-chain cyclization reaction solution

[0249] Component volume 10X reaction buffer 2μL CircLigase 1μL 50mM MnCl2 1μL 1mM ATP 0.5μL

[0250] 25. Add the prepared single-stranded circularization reaction solution to the PCR tube containing the denatured PCR product, mix thoroughly using a vortex mixer, and then centrifuge briefly to centrifuge the liquid to the bottom of the tube.

[0251] 26. Place the centrifuged PCR tube in a PCR instrument and perform the reaction according to the conditions in Table 17 below.

[0252] Table 17. Single-chain circularization reaction conditions

[0253] Temperature Time 105℃ Hot lid open 60℃ 1 hour 85℃ 10 minutes 4℃∞

[0254] 27. After the reaction is completed, centrifuge the PCR tube briefly and place it on ice before proceeding to the next step.

[0255] 28. Use the circularization kit from MGI (1000005260, MGI) and prepare the enzyme digestion reaction solution on ice in advance according to the recipe in Table 18 below.

[0256] Table 18. Enzyme digestion reaction solution

[0257] Components Single reaction volume Digestion buffer 1.4 μL Digestion enzyme 2.6 μL Total volume 4 μL

[0258] 29. Use a pipette to draw 4 μL of the prepared enzyme digestion reaction solution into the PCR tube. Vortex 3 times for 3 seconds each time. Centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0259] 30. Place the PCR tube on a PCR instrument and perform the reaction according to the conditions in Table 19 below.

[0260] Table 19. Enzymatic digestion reaction conditions

[0261] Temperature Time 75℃ Hot lid open 37℃ 30 minutes 4℃∞

[0262] 31. After the digestion reaction is completed, collect the reaction solution to the bottom of the tube by instant centrifugation.

[0263] 32. Immediately add 6 μL of digestion stop buffer (1000005260, MGI) to the PCR tube, vortex 3 times for 3 seconds each time, centrifuge briefly to collect the reaction solution at the bottom of the tube, and transfer all the reaction solution to a new 1.5 mL centrifuge tube.

[0264] 33. Then add 72 μL of purified magnetic beads to the 1.5 mL centrifuge tube, pipette 10 times to mix thoroughly, and incubate at room temperature for 5 minutes.

[0265] 34. After brief centrifugation, place the 1.5 mL centrifuge tube on a magnetic stand and let it stand for 5 minutes until the liquid becomes clear. Pipette out the supernatant and discard it.

[0266] 35. Add 200 μL of 75% ethanol to the 1.5 mL centrifuge tube and let it sit for 30 seconds to wash the magnetic beads. Repeat the washing step once.

[0267] 36. Try to drain the liquid in the centrifuge tube. If there is a small amount of liquid remaining on the wall of the tube, you can centrifuge the 1.5mL centrifuge tube briefly. After separation on the magnetic stand, use a small-scale pipette to drain the liquid at the bottom of the tube.

[0268] 37. Keep the 1.5 mL centrifuge tube fixed on the magnetic rack, open the centrifuge tube cap, and dry it at room temperature until the surface of the magnetic beads is non-reflective and free of cracks.

[0269] Remove the 1.5 mL centrifuge tube from the magnetic stand and add 21 μL of molecular-grade water to elute the DNA. Pipette gently 10 times to mix thoroughly, then incubate at room temperature for 5 minutes.

[0270] 39. After the brief centrifugation, place the 1.5 mL centrifuge tube on a magnetic stand and let it stand for 5 minutes until the liquid becomes clear. Then transfer 20 μL of the supernatant to a new 1.5 mL centrifuge tube.

[0271] 40. Use the fluorescence quantitative kit ( ssDNA Assay Kit) and quantify the purified products after enzyme digestion according to the operating instructions of the quantification kit.

[0272] 41. Use the MGISEQ-2000 High-Throughput Sequencing Reagent Set (PE100) (MGI, Cat. No. 1000012536) to prepare DNA nanoballs (DNBs) according to the instructions.

[0273] 42. Use the fluorescence quantitative kit ( ssDNA Assay Kit) and quantify DNB according to the operating instructions of the quantification kit.

[0274] 3. Experimental Results

[0275] The experimental results are shown in Table 20. As can be seen from this table, the product concentration after linear extension and purification was 7.2 ng / μL. After single-stranded circularization of the extension product and linear DNA digestion, the final single-stranded circular DNA library concentration was 1.6 ng / μL. 8 ng of the single-stranded circular library was used to prepare DNA nanoballs (DNBs) unique to the DNBSEQ platform, resulting in a DNB concentration of 20.5 ng / μL, which met the standard and confirmed that the single-stranded circular library was qualified.

[0276] Table 20. Experimental results

[0277]

Claims

1. A method for constructing a single-stranded nucleic acid circular library, comprising the following steps: 1) providing a single-stranded nucleic acid template, such as a single-stranded DNA template or a single-stranded RNA template; 2) forming a partially double-stranded linker element between the linker molecule and the auxiliary chain, wherein the 5' end portion of the auxiliary chain is reverse complementary to the 5' end portion of the linker molecule, and the 3' end of the auxiliary chain has a random base sequence that is hybridized and complementary to the 3' end portion of the single-stranded nucleic acid template; 3) ligating the linker molecule in the linker element to the 3' end of the single-stranded nucleic acid template using a ligase, thereby obtaining a nucleic acid fragment with a partial double strand; 4) Performing a single-stranded circularization reaction on the product in step 3) to obtain a single-stranded nucleic acid circular library.

2. The library construction method according to claim 1, wherein The adapter molecule comprises at least one sequencing primer binding sequence or a combination of multiple sequencing primer binding sequences.

3. The library construction method according to claim 1 or 2, wherein: When the single-stranded nucleic acid template is a single-stranded RNA template, the library construction method further includes step 3') between steps 3) and 4): reverse transcribing the single-stranded RNA template using an extension primer complementary to the 3' end sequence of the adapter molecule and a reverse transcriptase.

4. The library construction method according to any one of claims 1 to 2, wherein: The library construction method further comprises step 3") before step 4): performing a linear extension reaction on the partially double-stranded nucleic acid fragment obtained in step 3) using an extension primer, wherein the extension primer is reverse complementary to the 3' end sequence of the adapter molecule.

5. The library construction method according to claim 3 or 4, wherein: The adapter molecule comprises a sequencing primer binding sequence, the extension primer comprises a sequence reversely complementary to the sequencing primer binding sequence at the 3' end, and another sequencing primer binding sequence at the 5' end.

6. The library construction method according to any one of claims 1 to 5, wherein: The 5' end of the adapter molecule and / or the extension primer is phosphorylated, and the 3' end of the adapter molecule and / or the 3' end of the auxiliary chain is modified and blocked.

7. A method for constructing a single-stranded nucleic acid circular library, comprising the following steps: 1) providing a single-stranded DNA template as a single-stranded nucleic acid template; 2) forming a partially double-stranded linker element between the linker molecule and the auxiliary chain, wherein the 3' end portion of the auxiliary chain is reverse complementary to the 3' end portion of the linker molecule, and the 5' end of the auxiliary chain has a random base sequence that is hybridized and complementary to the 5' end portion of the single-stranded nucleic acid template; 3) ligating the linker molecule in the linker element to the 5' end of the single-stranded nucleic acid template using a ligase, thereby obtaining a nucleic acid fragment with a partial double strand; 4) Performing a single-stranded circularization reaction on the product obtained in step 3) to obtain a single-stranded nucleic acid circular library.

8. The library construction method according to claim 7, wherein: The adapter molecule comprises a combination of multiple sequencing primer binding sequences.

9. The library construction method according to claim 7 or 8, wherein: The 5' end of the linker molecule is phosphorylated and the 3' end of the auxiliary chain is modified and blocked.

10. The library construction method according to claim 6 or 9, wherein: The modifications used for blocking include phosphorylation modification, C3-spacer modification, amino modification and C6 Spacer modification.

11. The library construction method according to any one of claims 1 to 10, wherein: The length of the random base sequence is 6 to 12 nucleotides, preferably 12 nucleotides.

12. The library construction method according to any one of claims 1 to 11, wherein: The library construction method further comprises: after the single-stranded circularization reaction in step 4), digesting the uncircularized single-stranded nucleic acid using a linear digestion enzyme.

13. The library construction method according to any one of claims 1 to 12, wherein: The adapter molecule and / or the extension primer further comprises a tag sequence, wherein the tag sequence comprises a sample tag for distinguishing the source of a sample or a unique molecular tag for distinguishing a nucleic acid molecule. 14 . The method according to claim 13 , wherein the tag sequence is located between the multiple sequencing primer binding sequences of the adapter molecule.

15. The library construction method according to any one of claims 1 to 14, wherein: The single-stranded nucleic acid template comes from genomic DNA fragments, RNA fragments obtained by shearing the extracted total RNA, extracellular free nucleic acids, degraded nucleic acids extracted from degraded biological samples (such as formalin-fixed (FFPE) and paraffin-embedded biological tissue samples, forensic samples and paleontological fossils), or nucleic acids treated with sulfite.

16. A method for sequencing a single-stranded circular nucleic acid, the method comprising: a) performing the method for constructing a single-stranded nucleic acid circular library according to any one of claims 1 to 15 to obtain a single-stranded nucleic acid circular library; b) performing an amplification reaction on the single-stranded nucleic acid circular library obtained in step a) and sequencing to obtain sequencing data.

17. A kit for constructing a single-stranded nucleic acid circular library, the kit comprising: (1) an adapter molecule, for example, the adapter molecule comprises at least one sequencing primer binding sequence or a combination of multiple sequencing primer binding sequences; (2) an auxiliary chain, one end of which has a sequence that is reverse complementary to a portion of the sequence at the connecting end of the linker molecule, and the other end of which has a random base sequence that is complementary to the connecting end of the single-stranded nucleic acid template, for example, a random base sequence of 6 to 12 nucleotides in length, preferably 12 nucleotides in length; (3) Ligase, including: RNA ligase such as T4 RNA ligase 2, and DNA ligase such as T4 DNA ligase, T3 DNA ligase or Taq DNA ligase, preferably T4 DNA ligase or T4 RNA ligase 2; (4) An optional extension primer, wherein the extension primer comprises a sequence that is reverse complementary to the adapter molecule. Optionally, the extension primer further comprises a sequencing primer binding sequence, and the sequence that is reverse complementary to the adapter molecule is located at the 3' end of the extension primer, and the sequencing primer binding sequence is located at the 5' end of the extension primer.

18. The kit according to claim 17, wherein The 5' end of the adapter molecule and the 5' end of the extension primer are phosphorylated and modified, and the 3' end of the auxiliary chain is modified and blocked; optionally, the 3' end of the adapter molecule is modified and blocked.

19. The kit according to claim 18, wherein The modifications include phosphorylation modification, C3-spacer modification, amino modification and C6 Spacer modification.

20. The kit according to any one of claims 17 to 19, wherein The adapter molecule and / or the extension primer further comprises a tag sequence, wherein the tag sequence comprises a sample tag for distinguishing the source of a sample or a unique molecular tag for distinguishing a nucleic acid molecule.

21. The kit according to claim 20, wherein The tag sequence is located between the multiple sequencing primer binding sequences of the adapter molecule.

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