WO2023240611A1 - Library construction and sequencing method for single-stranded nucleic acid cyclic library - Google Patents

Abstract

The present invention belongs to the field of gene sequencing, and particularly relates to a library construction and sequencing method for a single-stranded nucleic acid cyclic library. In the method, a sequencing primer binding sequence is combined in a linker molecule or the sequencing primer binding sequence is separately contained in the linker molecule and an extension reaction primer, so that reaction steps for connecting a sequencing linker are reduced, the loss of a nucleic acid template is reduced, the linker connection efficiency is improved, the utilization rate of an original nucleic acid template is improved, and rapid library construction and sequencing of various nucleic acid samples are achieved.

Description

Single-stranded nucleic acid circular library construction and sequencing methods Technical field

The invention belongs to the field of gene sequencing. More specifically, the invention relates to a single-stranded nucleic acid circular library construction and sequencing method.

Background technique

At present, the common target for DNA sequencing library construction is complete genomic DNA with sufficient starting amount. The most conventional double-stranded DNA is usually used for library construction. The general steps include breaking the DNA into fragments of the required size and performing end repair. Afterwards, double-stranded adapters are connected to both ends of the DNA fragment template through 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 the samples usually suffers from severe degradation, and the DNA extracted from the samples is often severely degraded. The amount of DNA will also be very low. These DNA molecules usually contain both double-stranded DNA molecules and single-stranded DNA molecules, or there are single-strand breaks in the double-stranded DNA molecules. In addition, in methylation sequencing, DNA will be severely damaged after bisulfite conversion, resulting in the loss of most DNA molecules, and the DNA will be broken into short fragments and remain in a single-stranded state. In addition, the extracellular free DNA in plasma or other body fluid samples is itself a short fragment of DNA, and the DNA content is also very low.

For the above-mentioned special samples, it is not suitable to use the common double-stranded linker ligation library construction method, because the conventional library construction method can only target complete double-stranded DNA molecules, and cannot or rarely remove complete double-stranded DNA molecules. All other DNA template molecules are added with adapters, resulting in the loss of a large number of original DNA template molecules. The single-stranded library construction technology can utilize these single-stranded DNA template molecules, add linkers to these single-stranded DNA molecules, and build the library, thereby minimizing the loss of these irregular double-stranded DNA template molecules and maximizing the It improves the utilization rate of DNA template molecules to a certain extent, thereby improving the efficiency of library construction and improving the effective data volume and data quality of sequencing. In addition, single-strand library construction technology can also better use the above-mentioned special samples for corresponding application testing, such as liquid biopsy for extracellular cell-free DNA (cfDNA) methylation sequencing, genomic research of ancient biological DNA, forensic identification, and Tumor detection of FFPE samples, etc.

Existing single-stranded library construction technologies mainly include traditional single-stranded library construction methods, Swift's Adaptase single-stranded library construction technology, random primer plus adapter method, terminal transferase plus adapter method, and SPALT (Splinted adapter tagging) technology.

Traditional single-stranded library construction method: directly connect the single-stranded sequencing adapter to the single-stranded DNA template molecule through T4 RNA ligase. Generally, T4 RNA ligase 2 (truncated type) is used to catalyze 5' end pre-adenylation. The DNA adapter is connected to the 3' end of the single-stranded DNA template, and the 3' end of the adapter is blocked to reduce the generation of adapter dimers. After the obtained product is purified, a 5' end adapter and T4 RNA ligase are added to connect the 5' end adapter to the single-stranded DNA template that has been added with a 3' end adapter. After adding connectors at both ends, PCR amplification is performed. If truncated connectors were connected before, PCR also has the function of completing the remaining connectors.

Swift's Adaptase single-stranded library construction technology first uses a 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 that binds to the 3' end adapter primer for amplification. , convert the single-stranded DNA template into double-stranded DNA, and then add a 5'-end sequencing adapter to the 5' end of the newly synthesized strand through a conventional adapter ligation reaction to complete the construction of the library.

The single-stranded library construction technology of random primers uses 6nt to 8nt random primers to combine with a 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 strand to complete the process. Library construction.

The single-stranded library construction technology using terminal transferase is similar to the single-stranded library construction technology with random primers. First, terminal transferase is used to add continuous bases to the 3' end of the single-stranded DNA template, and then the continuous sequence is used as a primer combination. Position, extend, convert the single-stranded DNA template into double-stranded, and then add a 5'-end sequencing adapter to the 5' end of the newly synthesized strand to complete the construction of the library.

SPALT (Splinted adapter tagging) technology adds a double-stranded adapter to the 3' end of the single-stranded DNA template. The double-stranded adapter contains a section of random bases for complementary binding to single-stranded DNA. Then a double-stranded adapter containing random bases is added to the 5' end of the single-stranded template DNA. After adding adapters to both ends of the single strand, PCR amplification is performed to complete the construction of a single-stranded library.

In addition, if the above methods are to eventually build a single-stranded circular library, they all need to further denature the single strand, and then use DNA ligase and oligonucleotide chains with complementary connectors at both ends to perform circular ligation.

From the above, we can know that the traditional single-stranded library construction method uses T4 RNA ligase 2 (truncated type), which requires a 5'-end pre-adenylation modified DNA adapter, and the 5'-end pre-adenylation modification is expensive. , and it is difficult to synthesize, and only a few manufacturers can produce it, resulting in high costs. Furthermore, it is necessary to connect adapters at both ends of the DNA template and 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 all connect adapters at the 3' end and 5' end at both ends of the DNA template. Only after adding adapters at both ends can Perform PCR amplification to amplify the template. These increase the number of experimental steps, lead to the loss of the original DNA template, and reduce the utilization rate of the DNA template.

Therefore, there is an urgent need in this field for a method for constructing a single-stranded nucleic acid circular library. This method does not require adding adapters to both ends of the single-stranded nucleic acid template, nor does it require the use of costly special end modifications, thus reducing the number of reactions. This step also reduces costs, reduces the loss of nucleic acid templates, increases the efficiency of adapter ligation, and improves the utilization of original nucleic acid templates.

Contents of the invention

As mentioned above, the existing single-stranded nucleic acid circular library construction methods require connecting adapters to both ends of the nucleic acid template. PCR amplification can only be performed after adapters are added to both ends, which reduces the experimental steps and costs. increase, which also leads to the loss of the original nucleic acid template and reduces the utilization rate of the nucleic acid template. Therefore, there is an urgent need in this field for a method for constructing a single-stranded nucleic acid circular library that does not require adding adapters to both ends of the single-stranded nucleic acid template, nor does it require the use of costly special end modifications. Rapid library construction and sequencing of a variety of nucleic acid samples, especially short nucleic acid fragments, extracellular free nucleic acids, degraded nucleic acids and other special nucleic acid samples.

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

1) Provide single-stranded nucleic acid template;

2) Form a linker element with a partial double strand between the linker molecule and the auxiliary chain, wherein the 5' end of the linker molecule is phosphorylated and modified, the 3' end of the linker molecule is modified and blocked, and the auxiliary chain's The 5' end partial sequence is reverse complementary to the 5' end partial sequence of the adapter molecule, and the 3' end of the auxiliary strand has a random base sequence that is hybridized and complementary to the 3' end partial sequence of the single-stranded nucleic acid template. ;

3) Connect the linker molecule in the linker element to the 3' end of the single-stranded nucleic acid template through a ligase, thereby obtaining a nucleic acid fragment with a partial double-stranded structure;

4) Perform a single-strand cyclization reaction on the product in step 3) to obtain a single-stranded nucleic acid circular library.

In a second aspect, the present invention also provides a method for constructing a single-stranded nucleic acid circular library, including the following steps:

1) Provide single-stranded nucleic acid template;

2) Form a linker element with a partial double strand between the linker molecule and the auxiliary chain, wherein the 5' end of the linker molecule is phosphorylated, and the 3' end partial sequence of the auxiliary chain is consistent with the 3' end sequence of the linker molecule. The 'end partial sequence is reverse complementary, and the 5' end of the auxiliary strand has a random base sequence that is hybridized and complementary to the 5' end partial sequence of the single-stranded nucleic acid template;

3) Connect the linker molecule in the linker element to the 5' end of the single-stranded nucleic acid template through a ligase, thereby obtaining a nucleic acid fragment with a partial double-stranded structure;

4) Perform a single-strand cyclization reaction on the product obtained in step 3) to obtain a single-stranded nucleic acid circular library.

In a third aspect, the present invention also provides a method for sequencing single-stranded circular DNA, the method comprising:

a) Execute 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;

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

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

(1) Linker molecule, for example, the linker molecule includes at least one sequencing primer binding sequence or a combination of multiple sequencing primer binding sequences;

(2) An auxiliary strand. One end of the auxiliary strand has a sequence that is reverse complementary to the partial sequence of the connecting end of the adapter molecule, and the other end of the auxiliary strand has random bases that are hybridized and complementary to the connecting end of the single-stranded nucleic acid template. sequence;

(3) Ligase.

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

1) By combining multiple sequencing primer binding sequences in an adapter molecule or including multiple sequencing primer binding sequences in an adapter molecule and an extension reaction primer respectively, the reaction steps for connecting sequencing adapters are reduced and the loss of nucleic acid templates is reduced. , increasing the efficiency of adapter ligation and improving the utilization of the original nucleic acid template;

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

3) Achieve rapid library construction and sequencing of a variety of nucleic acid samples, especially short nucleic acid fragments, extracellular free nucleic acids, degraded nucleic acids and other special nucleic acid samples.

Description of the drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the drawings needed to be used in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some of the drawings of the present invention. Embodiments: For those of ordinary skill in the art, other embodiments can be obtained based on these drawings without exerting creative efforts.

Figure 1 is a schematic flow chart of one embodiment of a rapid library construction method for a single-stranded DNA circular library. In the library construction method shown in the figure, the adapter molecule contains a combination of two sequencing primer binding sequences, and the adapter molecule is connected at the 3' end of the single-stranded DNA template.

Figure 2 is a flow diagram of another embodiment of a rapid library construction method for 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.

Figure 3 is a schematic diagram of the position 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.

Figure 4 is a schematic flow chart of an embodiment of a rapid library construction method for a single-stranded DNA circular library. In the library construction method shown in the figure, the adapter molecule contains a combination of two sequencing primer binding sequences, and the adapter molecule is connected at the 5' end of the single-stranded DNA template.

Detailed ways

In order to better explain the purpose, technical solutions and advantages of the present invention, the present invention will be described clearly and completely below in conjunction with the embodiments and drawings of the present invention. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments in the present invention, all other embodiments that can be obtained by those of ordinary skill in the art fall within the scope of protection of the present invention.

As mentioned above, the existing methods of constructing single-stranded nucleic acid circular libraries require connecting adapters at both ends of the nucleic acid template, which increases the experimental steps and costs. It also leads to the loss of the original nucleic acid template and reduces the cost of the nucleic acid template. utilization rate.

Therefore, the object of the present invention is to provide a new method for constructing a single-stranded nucleic acid circular library. The inventors optimized and improved the existing single-stranded library construction technology by combining multiple sequencing primer binding sequences in the adapter molecule or including the multiple sequencing primer binding sequences in the adapter molecule and extension primer respectively, so that the A linker element with partial double strands is formed between the linker molecule and the auxiliary strand and is connected to the end (3' end or 5' end) of the single-stranded nucleic acid template through a ligase, thereby obtaining a nucleic acid fragment with partial double strands, as appropriate. The extension primer is used to perform an extension reaction, and finally the obtained product is subjected to a single-strand cyclization reaction, thereby completing the present invention.

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

1) Provide single-stranded nucleic acid template;

2) Form a linker element with a partial double strand between the linker molecule and the auxiliary chain, wherein the 5' end partial sequence of the auxiliary strand is reverse complementary to the 5' end partial sequence of the linker molecule, and the auxiliary chain The 3' end of the chain has a random base sequence that is hybridized and complementary to the 3' end partial sequence of the single-stranded nucleic acid template;

3) Connect the linker molecule in the linker element to the 3' end of the single-stranded nucleic acid template through a ligase, thereby obtaining a nucleic acid fragment with a partial double-stranded structure;

4) Perform a single-strand cyclization reaction on the product in step 3) to obtain a single-stranded nucleic acid circular library.

In a second aspect, the present invention also provides a method for constructing a single-stranded nucleic acid circular library, including the following steps:

1) Provide single-stranded nucleic acid template;

2) Form a linker element with a partial double strand between the linker molecule and the auxiliary chain, wherein the 3' end partial sequence of the auxiliary strand is reverse complementary to the 3' end partial sequence of the linker molecule, and the auxiliary chain The 5' end of the chain has a random base sequence that is hybridized and complementary to the 5' end partial sequence of the single-stranded nucleic acid template;

3) Connect the linker molecule in the linker element to the 5' end of the single-stranded nucleic acid template through a ligase, thereby obtaining a nucleic acid fragment with a partial double-stranded structure;

4) Perform a single-strand cyclization reaction on the product obtained in step 3) to obtain a single-stranded nucleic acid circular library.

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

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

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 with a size of 100 bp to 500 bp obtained by interrupting the complete genomic DNA and performing fragment screening. It is an RNA fragment obtained by fragmenting the extracted total RNA (including mRNA, IncRNA and small RNA, etc.). It can be extracted extracellular free nucleic acid, or it can be obtained from various degraded biological samples such as formalin-fixed organisms. Degraded nucleic acids extracted from tissue samples, paraffin-embedded biological tissue samples, forensic samples or paleontological fossils can also be DNA fragments after complete genomic DNA fragmentation or extracellular free nucleic acids that have been purified and recovered after sulfite treatment. Nucleic acid is not limited to this. Therefore, in one embodiment, the single-stranded nucleic acid template can be a DNA fragment obtained by fragmentation of denatured intact genomic DNA, an RNA fragment obtained by fragmenting the extracted total RNA, extracellular free nucleic acid, Degraded nucleic acid extracted from degraded biological samples, or 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 mentioned in the background art, the nucleic acids extracted from these samples are usually in low amounts, degraded, and contain both double-stranded and single-stranded nucleic acid molecules, making it impossible to use conventional library construction methods that can only utilize intact double-stranded nucleic acid molecules. . The library construction method of the present invention can use single-stranded nucleic acid templates to construct the library, thereby avoiding the loss of irregular double-stranded nucleic acid template molecules to the greatest extent, 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.

In addition, it can be 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. Double-stranded nucleic acids can be denatured using conventional denaturation methods in the art. For example, the DNA sample can be placed at 92°C to 98°C for 3-10 minutes, thereby allowing the double-stranded DNA to melt into single-stranded DNA. In addition, in order to prevent the renaturation of the melted single-stranded DNA, the sample needs to be cooled down immediately after denaturation, for example, by placing the sample on ice.

In step 2), a linker element with partial double strands is formed between the linker molecule and the auxiliary chain.

The linker molecule can be connected to the 5' end or the 3' end of the single-stranded nucleic acid template, depending on the situation. For example, when the amount of nucleic acid template is sufficient and PCR amplification is not required, it is feasible for the adapter molecule to be connected to the 5' end or 3' end of the single-stranded nucleic acid template; but when the amount of nucleic acid template is insufficient and therefore PCR amplification is required In this case, the adapter molecule needs to be connected to the 3' end of the single-stranded nucleic acid template to achieve PCR amplification of the single-stranded nucleic acid template.

In one embodiment, the 5' end partial sequence of the auxiliary strand is reverse complementary to the 5' end partial sequence of the adapter molecule, and the 3' end of the auxiliary strand has a 3' end sequence with the single-stranded nucleic acid template. The 'end partial sequence hybridizes to a complementary random base sequence. In this case, the adapter molecule is connected to the 3' end of the single-stranded nucleic acid template, and subsequent PCR amplification can be determined based on the amount of template.

In another embodiment, the 3' end partial sequence of the auxiliary strand is reverse complementary to the 3' end partial sequence of the adapter molecule, and the 5' end of the auxiliary strand has a sequence with the single-stranded nucleic acid template. The 5' end partial sequence hybridizes to a complementary random base sequence. In this case, the adapter molecule is attached to the 5' end of the single-stranded nucleic acid template. Since the single-stranded nucleic acid template cannot be further PCR amplified in this case, it is only suitable for situations where the amount of template is sufficient.

In one embodiment, the 5' end of the linker molecule is modified by phosphorylation, and the 3' end of the linker molecule is modified and blocked. The purpose of phosphorylating the 5' end of the adapter molecule is to enable the adapter molecule to undergo a ligation reaction 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 is to avoid self-ligation and non-specific amplification between the adapters, which greatly improves the construction efficiency. The utilization rate of templates in the library improves the specificity of the library. In one embodiment, the modifications for blocking include phosphorylation modifications, C3-spacer modifications, amino modifications and C6 Spacer modifications. In one embodiment, the 3' end of the linker molecule has a reversible blocking group. As used herein, "reversible blocking group" refers to a blocking group that can subsequently be reversibly removed from the linker molecule. In the case of subsequent PCR amplification, since the content of the original nucleic acid template becomes relatively low, the final cyclization reaction can be performed directly without removing the blocking group on the adapter molecule; and in the case of subsequent PCR amplification, the In this case, it is preferable to remove the blocking group on the linker molecule before performing the final cyclization reaction. In addition, when the linker molecule is connected to the 5' end of the single-stranded nucleic acid template, the 3' end of the linker molecule is not blocked in order to connect to the single-stranded nucleic acid template.

The library construction method of the present invention involves the use of auxiliary chains. In order to connect the auxiliary nucleic acid template and the adapter molecule, one end of the auxiliary strand needs to have a random base sequence that is 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 inverse to the partial sequence of the connected end of the adapter molecule. To complement each other. More specifically, when 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 strand is reverse complementary to the partial sequence at the 5' end of the adapter molecule, and the 3' end of the auxiliary strand has A random base sequence that is complementary to the 3' end of the nucleic acid template; when 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 strand is reverse complementary to the partial sequence at the 3' end of the adapter molecule. , and the 5' end of the auxiliary strand has a random base sequence that is complementary to the 5' end of the nucleic acid template. Through this structure, a linker element with partial double strands can be formed between the linker molecule and the auxiliary strand, and then a ligase can be used to connect the single-stranded nucleic acid template and the linker element together to form a nucleic acid with partial double strands. fragment.

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 blocked by modification, and the modifications include phosphorylation modification, C3-spacer modification, amino modification, C6 Spacer modification, etc., to avoid self-connection and interference between the auxiliary chains. Non-specific extension using the auxiliary strand as a primer.

In one embodiment, the sequence portion of the auxiliary strand that is reverse complementary to the linker molecule may 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.

In yet another embodiment, the complementary random base sequence of the auxiliary strand and the target DNA template may have 6-12 nucleotides, such as 6, 7, 8, 9, 10, 11 or 12 nucleotides. , preferably with 12 nucleotides.

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

As mentioned in the background section, in the prior art, two sequencing adapters need to be connected to the nucleic acid fragment respectively to achieve sequencing of the nucleic acid fragment. However, in the method of the present invention, two sequencing primer binding sequences used in the sequencing platform can be first connected into a linker molecule, and then the linker molecule is connected to the single-stranded nucleic acid template. At this time, the adapter molecule includes a combination of two sequencing primer binding sequences. In addition, in the method of the present invention, if the adapter molecule only contains a sequencing primer binding sequence, it is necessary to connect another sequencing primer binding sequence to the sequencing primer binding sequence and the single-stranded nucleic acid template through an extension reaction, See the description below for details.

In addition, in one embodiment, the adapter molecule may also include a tag sequence, which includes 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.

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

Those skilled in the art can select a specific ligase based on 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 may be an RNA ligase, including but not limited to T4 RNA ligase 2.

In one embodiment, 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): using the 3' end sequence of the adapter molecule Complementary extension primers and reverse transcriptase perform reverse transcription of the single-stranded RNA template.

First of all, it is important to note that step 3') is only applicable to the embodiment in which the adapter molecule is connected to the 3' end of the single-stranded nucleic acid template. Secondly, whether the method of the present invention includes step 3') depends on the type of single-stranded nucleic acid template used. Specifically, when using a single-stranded DNA template, step 3') does not need to exist, and when using a single-stranded RNA template, the RNA needs to be reverse transcribed into DNA before proceeding to the next step, and then proceed to the subsequent steps. Reverse transcription of RNA into DNA can use conventional reverse transcription techniques in the art, which will not be described further in this article.

In one embodiment, the library construction method of the present invention also includes step 3") before step 4): using an extension primer to perform a linear extension reaction on the nucleic acid fragment with partial double strands obtained in step 3), the extension primer 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 can Including this step is particularly suitable for situations where the amount of nucleic acid samples is insufficient. The library construction method of the present invention may not include this step. At this time, it is particularly suitable for situations where the amount of nucleic acid samples is sufficient; and when the amount of nucleic acid samples is sufficient, , the adapter molecule can 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 step of extension reaction.

In order to perform a linear extension reaction, the 5' end of the extension primer needs to be reverse complementary to the 3' end partial sequence of the adapter molecule to ensure the smooth start of the extension reaction. In addition, in order to avoid unwanted self-ligation between extension primers, the 5’ end of the extension primer can be phosphorylated.

In addition, it can be understood that in the case of performing an extension reaction, the auxiliary strand can be directly used as the extension primer. Therefore, in one embodiment, the 3' end of the auxiliary chain has a reversible blocking group. In this way, when performing an extension reaction, the reversible blocking group at the 3' end of the auxiliary strand can be removed first, and then the extension reaction can be performed using the auxiliary strand as an extension primer without the need to introduce additional extension primers.

As a further embodiment, the adapter molecule may only include a sequencing primer binding sequence, the extension primer may include a sequence at the 3' end that is reverse complementary to the sequencing primer binding sequence, and at the 5' end Contains another sequencing primer binding sequence. It can be understood that in this case, the linear extension reaction of step 3") is necessary in order to completely introduce the sequencing primer binding sequence through the extension reaction. Specifically, in the method of the present invention, the method for The first sequencing primer binding sequence of the sequencing platform is connected to the nucleic acid template, and then an extension primer including a second sequencing primer binding sequence and a sequence reversely complementary to the 3' end of the first sequencing primer binding sequence is used to amplify the sequence through PCR. The second sequencing primer binding sequence is connected to the first sequencing primer binding sequence and the single-stranded nucleic acid template; the partial sequence of the first sequencing primer binding sequence for the sequencing platform can be connected to the nucleic acid template first, and then used An extension primer that includes the second sequencing primer binding sequence, the remaining sequence of the first sequencing primer binding sequence, and a sequence that is reverse complementary to the 3' end of the partial sequence of the first sequencing primer binding sequence amplifies the second sequencing primer through PCR. The remaining sequences of the sequencing primer binding sequence and the first sequencing primer binding sequence are connected together with the partial sequence of the first sequencing primer binding sequence and the single-stranded nucleic acid template; or the first sequencing primer binding sequence for the sequencing platform can be first connected , and a partial sequence of the second sequencing primer binding sequence is connected to the nucleic acid template, and then a sequence including 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 is used. The extension primer connects the remaining sequence of the second sequencing primer binding sequence with the first sequencing primer binding sequence, the partial sequence of the second sequencing primer binding sequence and the single-stranded nucleic acid template through PCR amplification.

In some embodiments, the extension primer may further comprise a tag sequence that includes 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 connected to an adapter molecule and a single-stranded nucleic acid template through an extension reaction.

In addition, the linear extension reaction can be carried out in a conventional manner known in the art, and the number of cycles can be adjusted according to the input amount of nucleic acid, generally the number of cycles is between 5 and 30.

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

In step 4), the DNA fragment with partial double strands obtained in step 3) or 3') or the linear extension product obtained in step 3") can be denatured first, and then the single-stranded cyclization reaction can be performed; Alternatively, denaturation treatment and single-strand cyclization reaction can be performed at the same time. Conventional denaturation methods in the art can be used to denature the formed double-stranded DNA. For example, DNA fragments or extension products with partial double-strands can be denatured at 92°C to 98 ℃ for 3-10 minutes to allow the double-stranded DNA to melt into single-stranded DNA. In addition, in order to prevent the single-stranded DNA obtained from melting from renaturation, the sample needs to be cooled down immediately after denaturation, for example, by placing the sample on ice. Above. Through this denaturation treatment, single-stranded DNA in which the single-stranded DNA template and the adapter molecule are connected together can be obtained.

In addition, before proceeding to the next step, the obtained product can be purified to screen out the target product and avoid the impact 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), such as magnetic bead purification.

It can be understood that after step 4), there may be a portion of single-stranded nucleic acid templates that have not been circularized. For the purpose of more reliable subsequent sequencing, these remaining single-stranded nucleic acid templates can be processed. Therefore, in one embodiment, the library construction method further includes: after cyclizing the single-stranded nucleic acid in step 4), using a linear digestion enzyme to digest the uncirculated single-stranded nucleic acid.

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

It should be noted that, at least for the method of the first or second aspect of the present invention, although each step is labeled with numbers 1, 2, etc., it should be understood that the number labeling here is only for the purpose of distinguishing each step. It is not intended to indicate the sequence of steps. Specifically, the steps included in the method of the present invention can be performed in any order, such as sequentially, simultaneously or in reverse order, as long as the method can be finally implemented. It is within the ability of those skilled in the art to adjust the sequence of steps of the method of the invention.

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

a) Execute 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;

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

For the specific details of the method for constructing the single-stranded nucleic acid circular library described in the first or second aspect of the present invention in step a), please refer to the above descriptions of these two aspects, and will not be repeated here. Repeat.

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

(1) Linker molecules;

(2) An auxiliary strand. One end of the auxiliary strand has a sequence that is reverse complementary to the partial sequence of the connecting end of the adapter molecule, and the other end of the auxiliary strand has random bases that are hybridized and complementary to the connecting end of the single-stranded nucleic acid template. sequence;

(3) Ligase.

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

In one embodiment, the length of the random base sequence comprised by the auxiliary strand may be 6-12 nucleotides, preferably 12 nucleotides.

In one embodiment, the ligase can be the 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.

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 includes a sequencing primer binding sequence, and a 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 extension primer the 5' end.

In one embodiment, the 5' end of the adapter molecule and the 5' end of the extension primer are modified by phosphorylation, and the 3' end of the auxiliary strand is modified and blocked. In an optional embodiment, the 3' end of the linker molecule is also modified and blocked. In one embodiment, the modifications for blocking include phosphorylation modifications, C3-spacer modifications, amino modifications and C6 Spacer modifications.

In one embodiment, the adapter molecule and/or the extension primer further comprises a tag sequence, which includes a sample tag for distinguishing the source of the sample or a unique molecular tag for distinguishing nucleic acid molecules.

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

It can be understood that, in addition to the above composition, the kit may also contain other reagents, such as polymers, buffers, nucleotide mixtures, and the like.

The linker molecules, auxiliary strands, ligases and extension primers in the kit of this aspect of the invention are the same or similar to the linker molecules, auxiliary strands, ligases and extension primers of the first, second and third aspects of the invention, and therefore no longer Elaborate further.

The present invention will be further described below through the following examples. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Example

Example 1. Construction and sequencing of human peripheral blood cell-free DNA library

1. Experimental materials

Cell-free DNA extracted from human peripheral blood plasma

2. Experimental steps

1. Take 5ng human peripheral blood plasma cell-free DNA into a 200μL PCR tube, and add enzyme-free water to make the total volume 45μL. Place the DNA on a PCR machine for denaturation (95°C-3 minutes), and then immediately place it on an ice box.

2. Prepare the adapter-auxiliary strand mixture: anneal and hybridize 20 μM adapter molecules (a combined adapter used to connect the 5' and 3' end sequencing adapters of the DNBSEQ sequencing platform together) and 20 μM auxiliary strand at a ratio of 1:1. , forming a linker element with partial double strands. The reaction conditions were: 95°C for 3 minutes, then cooled to 4°C, and the cooling rate of the PCR machine was set to 0.1°C/second.

Sequence of linker molecule:

5’Pho-AAGTCGGATCGTAGCCATGTCGTTCTGTGAGCCAAGGAGTTGATCGGACCTATTGTCTTCCTAAGACCGCTTGGCCTCCGACTT-3’Pho

Sequence of auxiliary chain:

5’-ACATGGCTACGATCCGACTTNNNNNNNNNNNN-3’C3-spacer

3. Use the T4 DNA ligase and ligation buffer from the MGI enzyme digestion DNA library preparation kit (1000005254, MGI) to connect the adapter and DNA template. Prepare the reaction solution on the ice box according to Table 1 below.

Table 1. Connector connection reaction solution

Components volume T4 DNA ligase 1.6μL ligation buffer 23.4μL Connector components 10μL

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

5. Place the centrifuged PCR tube on the PCR machine and react at 30°C for 30 minutes to perform the ligation reaction.

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

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

8. Add 200 μL of 75% ethanol to the 1.5 mL centrifuge tube and let stand for 30 seconds to wash the magnetic beads. Repeat the step of washing the magnetic beads once.

9. Try to absorb the liquid in the centrifuge tube as much as possible. When there is a small amount of liquid remaining on the tube wall, the 1.5mL centrifuge tube can be centrifuged instantaneously. After separation on the magnetic stand, use a small-volume pipette to absorb the liquid at the bottom of the tube.

10. Keep the 1.5mL centrifuge tube fixed on the magnetic stand, open the cap of the centrifuge tube, and dry it at room temperature until the surface of the magnetic beads is non-reflective and has no cracks.

11. Remove the 1.5 mL centrifuge tube from the magnetic stand, add 23 μL molecular grade water for DNA elution, gently pipet 10 times with a pipette to mix completely, and then incubate at room temperature for 5 minutes.

12. Centrifuge briefly, place the 1.5mL centrifuge tube on a magnetic stand, let it sit for 5 minutes until the liquid is clear, and then transfer 22μL supernatant to a new 200μL PCR tube.

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

Extension Primer-L: 5’Pho-AAGTTCGGAGGCCAAGCG-3’

Table 2. Extension reaction solution

Components volume PCR enzyme mix 25μL PCR Primer-L 3μL

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

15. Place the centrifuged PCR tube on the PCR machine and perform the reaction according to Table 3 below.

Table 3. Extension reaction conditions

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, pipet 10 times with a pipette to mix thoroughly, and incubate at room temperature for 5 minutes. .

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

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 step of washing the magnetic beads once.

19. Try to absorb the liquid in the centrifuge tube as much as possible. When there is a small amount of liquid remaining on the tube wall, the 1.5mL centrifuge tube can be centrifuged instantaneously. After separation on the magnetic stand, use a small-volume pipette to absorb the liquid at the bottom of the tube.

20. Keep the 1.5mL centrifuge tube fixed on the magnetic stand, open the cap of the centrifuge tube, and dry it at room temperature until the surface of the magnetic beads is non-reflective and has no cracks.

21. Remove the 1.5 mL centrifuge tube from the magnetic stand, add 18 μL of molecular grade water for DNA elution, gently pipet 10 times with a pipette to mix completely, and then incubate at room temperature for 5 minutes.

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

ssDNA Assay Kit)，按照定量试剂盒的操作说明取1μL的PCR产物进行定量。 23. Place the PCR tube on the PCR machine for denaturation (95℃_3 minutes), and then immediately place it on the ice box. Use a fluorescence quantification kit (

ssDNA Assay Kit), follow the instructions of the quantification kit to take 1 μL of PCR product for quantification.

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

Table 4. Single chain cyclization reaction solution

Components volume 10X reaction buffer 2μL CircLigase 1μL 50mM _MnCl2 1μL 1mM ATP 0.5μL

25. Add the prepared single-strand cyclization reaction solution to the PCR tube containing the denatured PCR product, mix thoroughly using a oscillating mixer, and then centrifuge briefly to centrifuge the liquid to the bottom of the tube.

26. Place the centrifuged PCR tube on the PCR machine and perform the reaction according to the conditions in Table 5 below.

Table 5. Single chain cyclization reaction conditions

temperature time 105℃ hot cover turn on 60℃ 1 hour 85℃ 10 minutes 4℃ ∞

27. After the reaction, centrifuge the PCR tube briefly and place it on ice, and immediately proceed to the next step of the reaction.

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

Table 6. Enzyme digestion reaction solution

Components single reaction volume Digestion buffer 1.4μL Digestive enzymes 2.6μL total capacity 4μL

29. Use a pipette to add 4 μL of the prepared enzyme digestion reaction solution into the PCR tube, vortex 3 times, 3 seconds each time, and briefly centrifuge to collect the reaction solution to the bottom of the tube.

30. Place the PCR tube on the PCR machine and perform the reaction according to the conditions in Table 7 below.

Table 7. Digestion reaction conditions

temperature time 75℃ hot cover turn on 37℃ 30 minutes 4℃ ∞

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

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

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

34. After instant centrifugation, place the 1.5mL centrifuge tube on a magnetic stand and let it sit for 5 minutes until the liquid becomes clear. Use a pipette to absorb the supernatant and discard it.

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 step of washing the magnetic beads once.

36. Try to absorb the liquid in the centrifuge tube as much as possible. When there is a small amount of liquid remaining on the tube wall, you can perform instant centrifugation of the 1.5mL centrifuge tube. After separation on the magnetic stand, use a small-volume pipette to absorb the liquid at the bottom of the tube.

37. Keep the 1.5mL centrifuge tube fixed on the magnetic stand, open the cap of the centrifuge tube, and dry it at room temperature until the surface of the magnetic beads is non-reflective and has no cracks.

38. Remove the 1.5 mL centrifuge tube from the magnetic stand, add 21 μL molecular grade water for DNA elution, gently pipet 10 times with a pipette to mix completely, and then incubate at room temperature for 5 minutes.

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

ssDNA Assay Kit)，按照定量试剂盒的操作说明对酶切消化纯化后产物进行定量。 40.Use fluorescence quantification kit (

ssDNA Assay Kit), follow the instructions of the quantitative kit to quantify the products after digestion and purification.

41. Use MGISEQ-2000 High-Throughput Sequencing Reagent Kit (PE100) (MGI, Cat. No. 1000012536) and follow the instructions to prepare DNA nanospheres (DNB).

ssDNA Assay Kit)，按照定量试剂盒的操作说明对DNB进行定量。 42.Use fluorescence quantification kit (

ssDNA Assay Kit), follow the instructions of the quantification kit to quantify DNB.

3. Experimental results

The experimental results are shown in Table 8. As can be seen from the table, the concentration of the product obtained after linear extension and purification is 6.5ng/μL. After single-strand circularization and linear DNA digestion of the extension product, the concentration of the final single-stranded circular DNA library is 1.4 ng/μL. 8ng of the single-stranded loop library was taken to prepare DNA nanospheres (DNB) unique to the DNBSEQ platform. The obtained DNB concentration was 18.4ng/μL, which met the standard and also indicated that the single-stranded loop library obtained was qualified.

Table 8. Experimental results

Example 2. Construction and sequencing of human peripheral blood cell-free DNA methylation library

1. Experimental materials

Cell-free DNA extracted from human peripheral blood plasma

2. Experimental steps

1. Take 5ng of human peripheral blood plasma cell-free DNA (cfDNA) into a 200μL PCR tube, with a total volume of 20μL.

2. Use EZ DNA Methylation-Gold Edition Kit (Zymo Research, Cat. No. D5005/D5006) to perform bisulfite treatment and purification of cfDNA.

3. Add 900μL nanofiltration water, 300μL M-dilution buffer and 50μL M-dissolution buffer into a tube of CT Conversion Reagent powder (centrifuge briefly before opening the cap), vortex frequently at room temperature for 10 minutes to complete CT Preparation of Conversion Reagent. The exposure of CT Conversion Reagent should be reduced, and it is best to prepare it for immediate use. 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-prepared CT Conversion Reagent should be preheated to 37°C before use. Vortex frequently for 10 minutes at room temperature before use.

4. Before opening the cap of M-washing buffer for the first time, add the correct volume of absolute ethanol as shown on the bottle label, and mix evenly before use. At room temperature, add the ingredients in Table 9 to a new 200 μL PCR tube.

Table 9. Reagents and samples for bisulfite treatment

Components volume CT Conversion Reagent 130μL cfDNA 20μL total amount 150μL

5. Place the 200 μL PCR tube on the PCR machine and perform the reaction according to the bisulfite treatment conditions in Table 10.

Table 10. Bisulfite treatment conditions

temperature time hot cover turn on 98℃ 10 minutes 64℃ 2.5 hours 4℃ ∞

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

7. Put the Zymo-Spin IC column into a 2mL collection tube, transfer the mixture to the Zymo-Spin IC column, centrifuge at 13000 rpm for 30 seconds, discard the waste liquid, and put the Zymo-Spin IC column back into the collection tube.

8. Add 100μL M-wash buffer to the Zymo-Spin IC column and centrifuge at 13000rpm for 30 seconds.

9. Add 200 μL M-desulfonation buffer to the Zymo-Spin IC column, quickly cap the tube tightly, incubate at room temperature for 15 to 20 minutes, centrifuge at 13000 rpm for 30 seconds, discard the waste liquid, and replace the Zymo-Spin IC column. Place back into the collection tube.

10. Add 200μL M-wash buffer to the Zymo-Spin IC column, centrifuge at 13000rpm for 30 seconds, discard the waste liquid, and put the Zymo-Spin IC column back into the collection tube.

11. Add 200μL M-wash buffer to the Zymo-Spin IC column, centrifuge at 13000rpm for 30 seconds, discard the waste liquid, and put the Zymo-Spin IC column back into the collection tube. Centrifuge idling for 30 seconds at 13000 rpm, discard the collection tube, use a pipette to absorb as much liquid as possible from the outer wall of the Zymo-Spin IC column, and place it in a new 1.5 mL centrifuge tube.

12. Open the cap of the Zymo-Spin IC column, dry it at room temperature for 2 minutes, and then place the Zymo-Spin IC column into a new 1.5mL centrifuge tube.

13. Slowly add 10 μL M-elution buffer to the center of the Zymo-Spin IC column filter membrane, let it stand for 1 minute, and centrifuge at 13000 rpm for 30 seconds. The purified bisulfite-treated purified product is collected in 1.5 mL. in a centrifuge tube.

14. Put all the bisulfite-treated and purified products into a new 200μL PCR tube, add molecular grade water to make the total volume to 20μL.

15. Subsequently, all experimental steps in Example 1 were performed on the 20 μL sample thus obtained.

ssDNA Assay Kit荧光定量试剂盒，按照定量试剂盒的操作说明对DNB进行定量。 16. Use the MGISEQ-2000 sequencer for sequencing, use the MGISEQ-2000 high-throughput sequencing reagent set (PE100) (MGI, Cat. No. 1000012536), and follow the instructions to prepare and sequence DNB. use

ssDNA Assay Kit Fluorescence Quantitative Kit, follow the instructions of the quantitative kit to quantify DNB.

3. Experimental results

a. Database construction results

The specific results are shown in Table 11. As can be seen from the table, after sulfite conversion of cfDNA and purification after linear extension, the concentration of the product obtained is 7.8ng/μL. After single-strand circularization and linear DNA digestion of the extension product, the final single The concentration of the circular DNA library was 2.3ng/μL. 8ng of the single-stranded loop library was taken to prepare DNA nanospheres (DNB) unique to the DNBSEQ platform. The obtained DNB concentration was 18.1ng/μL, which met the standard and also indicated that the single-stranded loop library obtained was qualified.

Table 11. Database construction results

b. Sequencing results

After the DNB prepared in Example 2 is sequenced on a machine, the conventional whole-genome methylation sequencing analysis process is used to analyze the obtained data, including filtering of low-quality data, comparison with the reference genome, and analysis of coverage and transformation. The rate and number of CG sites were statistically calculated, and the corresponding analysis results were obtained (Table 12).

Table 12. Sequencing results

Example 3. Construction and sequencing of human peripheral blood cell-free DNA library

1. Experimental materials

Cell-free DNA extracted from human peripheral blood plasma

2. Experimental steps

1. Take 5ng human peripheral blood plasma cell-free DNA into a 200μL PCR tube, and add enzyme-free water to make the total volume 45μL. Place the DNA on a PCR machine for denaturation (95°C-3 minutes), and then immediately place it on an ice box.

2. Prepare the adapter-auxiliary strand mixture: anneal and hybridize 20 μM adapter molecules (a combined adapter used to connect the 5' and 3' end sequencing adapters of the DNBSEQ sequencing platform together) and 20 μM auxiliary strand at a ratio of 1:1. , forming a linker element with partial double strands. The reaction conditions were: 95°C for 3 minutes, then cooled to 4°C, and the cooling rate of the PCR machine was set to 0.1°C/second.

Sequence of adapter molecule:

5’Pho-AAGTCGGATCGTAGCCATGTCGTTCTGTGAGCCAAGGAG-3’Pho

Sequence of auxiliary chain:

5’-ACATGGCTACGATCCGACTTNNNNNNNNNNNN-3’C3-spacer

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

Table 13. Connector connection reaction solution

Components volume T4 DNA ligase 1.6μL ligation buffer 23.4μL Connector components 10μL

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

5. Place the centrifuged PCR tube on the PCR machine and react at 30°C for 30 minutes to perform the ligation reaction.

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

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

8. Add 200 μL of 75% ethanol to the 1.5 mL centrifuge tube and let stand for 30 seconds to wash the magnetic beads. Repeat the step of washing the magnetic beads once.

9. Try to absorb the liquid in the centrifuge tube as much as possible. When there is a small amount of liquid remaining on the tube wall, the 1.5mL centrifuge tube can be centrifuged instantaneously. After separation on the magnetic stand, use a small-volume pipette to absorb the liquid at the bottom of the tube.

10. Keep the 1.5mL centrifuge tube fixed on the magnetic stand, open the cap of the centrifuge tube, and dry it at room temperature until the surface of the magnetic beads is non-reflective and has no cracks.

11. Remove the 1.5 mL centrifuge tube from the magnetic stand, add 23 μL molecular grade water for DNA elution, gently pipet 10 times with a pipette to mix completely, and then incubate at room temperature for 5 minutes.

12. Centrifuge briefly, place the 1.5mL centrifuge tube on a magnetic stand, let it sit for 5 minutes until the liquid is clear, and then transfer 22μL supernatant to a new 200μL PCR tube.

13. Use 20 μM Extension Primer-L and the PCR enzyme mixture in the MGI digested DNA library preparation kit (1000005254, MGI) to prepare the extension reaction solution according to Table 14 below.

Extension Primer-L: 5'-AAGTCGGAGGCCAAGCGGTCTTAGGAAGACAATAGGTCCGATCAACTCCTTGGCTCACAGAACGACATG-3'

Table 14. Extension reaction solution

Components volume PCR enzyme mix 25μL PCR Primer-L 3μL

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

15. Place the centrifuged PCR tube on the PCR machine and perform the reaction according to Table 15 below.

Table 15. Extension reaction conditions

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, pipet 10 times with a pipette to mix thoroughly, and incubate at room temperature for 5 minutes. .

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

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 step of washing the magnetic beads once.

19. Try to absorb the liquid in the centrifuge tube as much as possible. When there is a small amount of liquid remaining on the tube wall, the 1.5mL centrifuge tube can be centrifuged instantaneously. After separation on the magnetic stand, use a small-volume pipette to absorb the liquid at the bottom of the tube.

20. Keep the 1.5mL centrifuge tube fixed on the magnetic stand, open the cap of the centrifuge tube, and dry it at room temperature until the surface of the magnetic beads is non-reflective and has no cracks.

21. Remove the 1.5 mL centrifuge tube from the magnetic stand, add 18 μL of molecular grade water for DNA elution, gently pipet 10 times with a pipette to mix completely, and then incubate at room temperature for 5 minutes.

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

ssDNA Assay Kit)，按照定量试剂盒的操作说明取1μL的PCR产物进行定量。 23. Place the PCR tube on the PCR machine for denaturation (95℃_3 minutes), and then immediately place it on the ice box. Use a fluorescence quantification kit (

ssDNA Assay Kit), follow the instructions of the quantification kit to take 1 μL of PCR product for quantification.

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

Table 16. Single chain cyclization reaction solution

Components volume 10X reaction buffer 2μL CircLigase 1μL 50mM _MnCl2 1μL 1mM ATP 0.5μL

25. Add the prepared single-strand cyclization reaction solution to the PCR tube containing the denatured PCR product, mix thoroughly using a oscillating mixer, and then centrifuge briefly to centrifuge the liquid to the bottom of the tube.

26. Place the centrifuged PCR tube on the PCR machine and perform the reaction according to the conditions in Table 17 below.

Table 17. Single chain cyclization reaction conditions

temperature time 105℃ hot cover turn on 60℃ 1 hour 85℃ 10 minutes 4℃ ∞

27. After the reaction, centrifuge the PCR tube briefly and place it on ice, and immediately proceed to the next step of the reaction.

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

Table 18. Enzyme digestion reaction solution

Components single reaction volume Digestion buffer 1.4μL Digestive enzymes 2.6μL total capacity 4μL

29. Use a pipette to add 4 μL of the prepared enzyme digestion reaction solution into the PCR tube, vortex 3 times, 3 seconds each time, and briefly centrifuge to collect the reaction solution to the bottom of the tube.

30. Place the PCR tube on the PCR machine and perform the reaction according to the conditions in Table 19 below.

Table 19. Digestion reaction conditions

temperature time 75℃ hot cover turn on 37℃ 30 minutes 4℃ ∞

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

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

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

34. After instant centrifugation, place the 1.5mL centrifuge tube on a magnetic stand and let it sit for 5 minutes until the liquid becomes clear. Use a pipette to absorb the supernatant and discard it.

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 step of washing the magnetic beads once.

36. Try to absorb the liquid in the centrifuge tube as much as possible. When there is a small amount of liquid remaining on the tube wall, you can perform instant centrifugation of the 1.5mL centrifuge tube. After separation on the magnetic stand, use a small-volume pipette to absorb the liquid at the bottom of the tube.

37. Keep the 1.5mL centrifuge tube fixed on the magnetic stand, open the cap of the centrifuge tube, and dry it at room temperature until the surface of the magnetic beads is non-reflective and has no cracks.

38. Remove the 1.5 mL centrifuge tube from the magnetic stand, add 21 μL molecular grade water for DNA elution, gently pipet 10 times with a pipette to mix completely, and then incubate at room temperature for 5 minutes.

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

ssDNA Assay Kit)，按照定量试剂盒的操作说明对酶切消化纯化后产物进行定量。 40.Use fluorescence quantification kit (

ssDNA Assay Kit), follow the instructions of the quantitative kit to quantify the products after digestion and purification.

41. Use MGISEQ-2000 High-Throughput Sequencing Reagent Kit (PE100) (MGI, Cat. No. 1000012536) and follow the instructions to prepare DNA nanospheres (DNB).

ssDNA Assay Kit)，按照定量试剂盒的操作说明对DNB进行定量。 42.Use fluorescence quantification kit (

ssDNA Assay Kit), follow the instructions of the quantification kit to quantify DNB.

3. Experimental results

The experimental results are shown in Table 20. As can be seen from the table, the concentration of the product obtained after linear extension and purification is 7.2ng/μL. After single-strand circularization and linear DNA digestion of the extension product, the concentration of the final single-stranded circular DNA library is 1.6 ng/μL. 8ng of the single-stranded loop library was taken to prepare DNA nanospheres (DNB) unique to the DNBSEQ platform. The obtained DNB concentration was 20.5ng/μL, which met the standard and also indicated that the single-stranded loop library obtained was qualified.

Table 20. Experimental results

Claims (21)

A method for constructing a single-stranded nucleic acid circular library, including the following steps: 1) Provide single-stranded nucleic acid templates, such as single-stranded DNA templates or single-stranded RNA templates; 2) Form a linker element with a partial double strand between the linker molecule and the auxiliary chain, wherein the 5' end partial sequence of the auxiliary strand is reverse complementary to the 5' end partial sequence of the linker molecule, and the auxiliary chain The 3' end of the chain has a random base sequence that is hybridized and complementary to the 3' end partial sequence of the single-stranded nucleic acid template; 3) Connect the linker molecule in the linker element to the 3' end of the single-stranded nucleic acid template through a ligase, thereby obtaining a nucleic acid fragment with a partial double-stranded structure; 4) Perform a single-strand cyclization reaction on the product in step 3) to obtain a single-stranded nucleic acid circular library. 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. 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): The single-stranded RNA template is reverse transcribed using an extension primer complementary to the 3' end sequence of the adapter molecule and reverse transcriptase. The library construction method according to any one of claims 1-2, wherein the library construction method further includes step 3") before step 4): using an extension primer to pair the partial double-stranded DNA obtained in step 3) The nucleic acid fragment is subjected to a linear extension reaction, and the extension primer is reverse complementary to the 3' end sequence of the adapter molecule. The library construction method according to claim 3 or 4, wherein the adapter molecule includes a sequencing primer binding sequence, and the extension primer includes a sequence at the 3' end that is reverse complementary to the sequencing primer binding sequence, And contains another sequencing primer binding sequence at the 5' end. The library construction method according to any one of claims 1 to 5, wherein the 5' end of the linker molecule and/or the extension primer is phosphorylated, and the 3' end of the linker molecule and/or The 3' end of the auxiliary chain is modified and blocked. A method for constructing a single-stranded nucleic acid circular library, including the following steps: 1) Provide single-stranded DNA template as single-stranded nucleic acid template; 2) Form a linker element with a partial double strand between the linker molecule and the auxiliary chain, wherein the 3' end partial sequence of the auxiliary chain is reverse complementary to the 3' end partial sequence of the linker molecule, and the auxiliary chain The 5' end of the chain has a random base sequence that is hybridized and complementary to the 5' end partial sequence of the single-stranded nucleic acid template; 3) Connect the linker molecule in the linker element to the 5' end of the single-stranded nucleic acid template through a ligase, thereby obtaining a nucleic acid fragment with a partial double-stranded structure; 4) Perform a single-strand cyclization reaction on the product obtained in step 3) to obtain a single-stranded nucleic acid circular library. The library construction method according to claim 7, wherein the adapter molecule comprises a combination of multiple sequencing primer binding sequences. The library construction method according to claim 7 or 8, wherein the 5' end of the linker molecule is modified by phosphorylation, and the 3' end of the auxiliary chain is modified and blocked. The library construction method according to claim 6 or 9, wherein the modification used for blocking includes phosphorylation modification, C3-spacer modification, amino modification and C6 Spacer modification. 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. The library construction method according to any one of claims 1-11, wherein the library construction method further includes: after the single-chain cyclization reaction in step 4), using a linear digestion enzyme to digest the uncyclized single-chain Nucleic acids are digested. The library construction method according to any one of claims 1-12, wherein the adapter molecule and/or the extension primer further comprises a tag sequence, the tag sequence includes a sample tag for distinguishing the source of the sample or for Unique molecular tags that distinguish nucleic acid molecules. According to the method of claim 13, the tag sequence is located between multiple sequencing primer binding sequences of the adapter molecule. The library construction method according to any one of claims 1 to 14, wherein the single-stranded nucleic acid template is derived from genomic DNA fragments, RNA fragments obtained by fragmenting the extracted total RNA, extracellular free nucleic acids, and degradation products. Degraded nucleic acids extracted from biological samples (such as formalin-fixed (FFPE) and paraffin-embedded biological tissue samples, forensic samples and paleontological fossils), or nucleic acids treated with sulfite. A method for sequencing single-stranded circular nucleic acids, the method comprising: a) Execute the method for constructing a single-stranded nucleic acid circular library according to any one of claims 1-15 to obtain a single-stranded nucleic acid circular library; b) Perform amplification reaction and sequencing on the single-stranded nucleic acid circular library obtained in step a) to obtain sequencing data. A kit for constructing a single-stranded nucleic acid circular library, the kit includes: (1) Linker molecule, for example, the linker molecule includes at least one sequencing primer binding sequence or a combination of multiple sequencing primer binding sequences; (2) An auxiliary strand. One end of the auxiliary strand has a sequence that is reverse complementary to the partial sequence of the connecting end of the adapter molecule, and the other end of the auxiliary strand has random bases that are hybridized and complementary to the connecting end of the single-stranded nucleic acid template. Sequence, for example, a random base sequence of 6 to 12 nucleotides in length, preferably 12 nucleotides; (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) Optional extension primer. The extension primer includes a sequence that is reverse complementary to the adapter molecule. Optionally, the extension primer also includes a sequencing primer binding sequence that is reverse complementary to the adapter molecule. The complementary sequence 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. The kit according to claim 17, wherein the 5' end of the adapter molecule and the 5' end of the extension primer are modified by phosphorylation, and the 3' end of the auxiliary chain is modified and blocked; optionally , the 3' end of the linker molecule is modified and blocked. The kit according to claim 18, wherein the modification includes phosphorylation modification, C3-spacer modification, amino modification and C6 Spacer modification. 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, the tag sequence includes a sample tag for distinguishing the source of the sample or for Unique molecular tags that distinguish nucleic acid molecules. The kit according to claim 20, wherein the tag sequence is located between multiple sequencing primer binding sequences of the adapter molecule.

Images