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WO2023240611A1 - Construction de banque et procédé de séquençage d'une banque cyclique d'acide nucléique simple brin - Google Patents

Construction de banque et procédé de séquençage d'une banque cyclique d'acide nucléique simple brin Download PDF

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WO2023240611A1
WO2023240611A1 PCT/CN2022/099514 CN2022099514W WO2023240611A1 WO 2023240611 A1 WO2023240611 A1 WO 2023240611A1 CN 2022099514 W CN2022099514 W CN 2022099514W WO 2023240611 A1 WO2023240611 A1 WO 2023240611A1
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nucleic acid
sequence
stranded
library
stranded nucleic
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WO2023240611A9 (fr
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夏军
宋喆
孔格致
杨新
赵霞
张艳艳
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MGI Tech Co Ltd
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MGI Tech Co Ltd
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Priority to CN202280096181.5A priority Critical patent/CN119213142A/zh
Priority to PCT/CN2022/099514 priority patent/WO2023240611A1/fr
Publication of WO2023240611A1 publication Critical patent/WO2023240611A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • 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.
  • 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.
  • FFPE formalin-fixed
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • cfDNA extracellular cell-free DNA
  • 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.
  • 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.
  • 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.
  • 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 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.
  • 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.
  • SPALT Smooth adapter tagging
  • 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.
  • 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.
  • 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.
  • the present invention provides a method for constructing a single-stranded nucleic acid circular library, including the following steps:
  • step 4) Perform a single-strand cyclization reaction on the product in step 3) to obtain a single-stranded nucleic acid circular library.
  • the present invention also provides a method for constructing a single-stranded nucleic acid circular library, including the following steps:
  • step 4) Perform a single-strand cyclization reaction on the product obtained in step 3) to obtain a single-stranded nucleic acid circular library.
  • the present invention also provides a method for sequencing single-stranded circular DNA, the method comprising:
  • step b) Perform amplification reaction and sequencing on the single-stranded nucleic acid circular library product obtained in step a) to obtain sequencing data.
  • the present invention provides a kit for constructing a single-stranded nucleic acid circular library, the kit comprising:
  • Linker molecule for example, the linker molecule includes at least one sequencing primer binding sequence or a combination of multiple sequencing primer binding sequences;
  • 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;
  • Figure 1 is a schematic flow chart of one embodiment of a rapid library construction method for a single-stranded DNA circular library.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention provides a method for constructing a single-stranded nucleic acid circular library, including the following steps:
  • step 4) Perform a single-strand cyclization reaction on the product in step 3) to obtain a single-stranded nucleic acid circular library.
  • the present invention also provides a method for constructing a single-stranded nucleic acid circular library, including the following steps:
  • step 4) Perform a single-strand cyclization reaction on the product obtained in step 3) to obtain a single-stranded nucleic acid circular library.
  • 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.
  • 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.
  • the degraded biological samples may include formalin-fixed (FFPE) and paraffin-embedded biological tissue samples, forensic samples, and paleontological fossils.
  • FFPE formalin-fixed
  • 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.
  • 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.
  • 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.
  • 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.
  • the sample needs to be cooled down immediately after denaturation, for example, by placing the sample on ice.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the modifications for blocking include phosphorylation modifications, C3-spacer modifications, amino modifications and C6 Spacer modifications.
  • the 3' end of the linker molecule has a reversible blocking group.
  • reversible blocking group refers to a blocking group that can subsequently be reversibly removed from the linker molecule.
  • 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.
  • 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.
  • 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.
  • the 5' end of the auxiliary strand has a random base sequence that is complementary to the 5' end of the nucleic acid template.
  • 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.
  • 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.
  • 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.
  • 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.
  • the adapter molecule may comprise at least one sequencing primer binding sequence or a combination of multiple sequencing primer binding sequences.
  • two sequencing adapters need to be connected to the nucleic acid fragment respectively to achieve sequencing of the nucleic acid fragment.
  • 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.
  • the adapter molecule includes a combination of two sequencing primer binding sequences.
  • the adapter molecule 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.
  • 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.
  • the tag sequence may be located between multiple sequencing primer binding sequences in the adapter molecule.
  • 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.
  • 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.
  • the ligase may be an RNA ligase, including but not limited to T4 RNA ligase 2.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • this step is optional.
  • 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.
  • 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.
  • the 5' end of the extension primer 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.
  • the auxiliary strand in the case of performing an extension reaction, 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.
  • 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.
  • the linear extension reaction of step 3 is necessary in order to completely introduce the sequencing primer binding sequence through the extension reaction.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 °C for 3-10 minutes to allow the double-stranded DNA to melt into single-stranded DNA.
  • the sample 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.
  • 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.
  • 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.
  • 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.
  • the present invention provides a method for sequencing single-stranded circular DNA, the method comprising:
  • step b) Sequencing the single-stranded nucleic acid circular library product obtained in step a) to obtain sequencing data.
  • the present invention provides a kit for constructing a single-stranded nucleic acid circular library, the kit comprising:
  • 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;
  • the adapter molecule may comprise at least one sequencing primer binding sequence or a combination of multiple sequencing primer binding sequences.
  • the length of the random base sequence comprised by the auxiliary strand may be 6-12 nucleotides, preferably 12 nucleotides.
  • 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.
  • the ligase may be an RNA ligase, including but not limited to T4 RNA ligase 2.
  • the kit may further comprise an extension primer comprising a sequence that is reverse complementary to the adapter molecule.
  • 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.
  • 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.
  • the 3' end of the linker molecule is also modified and blocked.
  • the modifications for blocking include phosphorylation modifications, C3-spacer modifications, amino modifications and C6 Spacer modifications.
  • 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.
  • the tag sequence is located between multiple sequencing primer binding sequences of the adapter molecule.
  • the kit may also contain other reagents, such as polymers, buffers, nucleotide mixtures, and the like.
  • 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.
  • 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.
  • adapter-auxiliary strand mixture anneal and hybridize 20 ⁇ M adapter molecules (a combined adapter used to connect the 5' and 3' end sequencing adapters of the DNBSEQ sequencing platform together) and 20 ⁇ M 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.
  • 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.
  • 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.
  • CircLigase kit (CL4111K, epicentre) to circularize the obtained single-stranded DNA to form a single-stranded DNA circular library.
  • 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/ ⁇ L. 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/ ⁇ L. 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/ ⁇ L, which met the standard and also indicated that the single-stranded loop library obtained was qualified.
  • DNB DNA nanospheres
  • cfDNA human peripheral blood plasma cell-free DNA
  • 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.
  • Example 1 All experimental steps in Example 1 were performed on the 20 ⁇ L sample thus obtained.
  • 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.
  • PE100 high-throughput sequencing reagent set
  • ssDNA Assay Kit Fluorescence Quantitative Kit follow the instructions of the quantitative kit to quantify DNB.
  • Example 12 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).
  • adapter-auxiliary strand mixture anneal and hybridize 20 ⁇ M adapter molecules (a combined adapter used to connect the 5' and 3' end sequencing adapters of the DNBSEQ sequencing platform together) and 20 ⁇ M 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.
  • T4 DNA ligase and ligation buffer from MGI uses 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.
  • 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.
  • 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.
  • CircLigase kit (CL4111K, epicentre) to circularize the obtained single-stranded DNA to form a single-stranded DNA circular library.
  • the experimental results are shown in Table 20.
  • the concentration of the product obtained after linear extension and purification is 7.2ng/ ⁇ L.
  • the concentration of the final single-stranded circular DNA library is 1.6 ng/ ⁇ L.
  • 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/ ⁇ L, which met the standard and also indicated that the single-stranded loop library obtained was qualified.

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Abstract

La présente invention appartient au domaine du séquençage de gènes, et concerne plus particulièrement un procédé de construction de banque et de séquençage d'une banque cyclique d'acide nucléique simple brin. Selon le procédé, une séquence de liaison d'amorce de séquençage est combinée dans une molécule de liaison ou bien la séquence de liaison d'amorce de séquençage est contenue séparément dans la molécule de liaison et dans une amorce de réaction d'extension, afin de réduire les étapes de réaction pour la connexion d'une amorce de séquençage, de réduire la perte d'une matrice d'acide nucléique, d'améliorer l'efficacité de la connexion d'amorce, d'améliorer le taux d'utilisation d'une matrice d'acide nucléique initiale et d'obtenir une construction de banque et un séquençage rapides de divers échantillons d'acide nucléique.
PCT/CN2022/099514 2022-06-17 2022-06-17 Construction de banque et procédé de séquençage d'une banque cyclique d'acide nucléique simple brin Ceased WO2023240611A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117845339A (zh) * 2024-01-09 2024-04-09 广西壮族自治区人民医院 一种用于检测与目标基因座相互作用的dna片段的文库构建方法
CN120060439A (zh) * 2025-02-27 2025-05-30 江苏硕世生物科技股份有限公司 一种基于封闭引物去人源核酸的测序文库构建方法及应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN119614676A (zh) * 2025-02-11 2025-03-14 天津中合基因科技有限公司 一种短链ssDNA的测序文库构建方法、测序方法及应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015026853A2 (fr) * 2013-08-19 2015-02-26 Abbott Molecular Inc. Bibliothèques de séquençage de nouvelle génération
CN105400776A (zh) * 2014-09-12 2016-03-16 深圳华大基因科技有限公司 寡核苷酸接头及其在构建核酸测序单链环状文库中的应用
WO2016037416A1 (fr) * 2014-09-12 2016-03-17 深圳华大基因科技有限公司 Lieur vésiculaire et ses utilisations dans la construction et le séquençage d'une bibliothèque d'acides nucléiques
CN105714383A (zh) * 2014-12-22 2016-06-29 深圳华大基因研究院 一种基于分子反向探针的测序文库构建方法和试剂
CN105985945A (zh) * 2015-01-30 2016-10-05 深圳华大基因研究院 mRNA片段化方法及基于其构建测序文库的方法
WO2019149958A1 (fr) * 2018-02-05 2019-08-08 F. Hoffmann-La Roche Ag Génération de modèles d'adn circulaires à simple brin pour une molécule unique
CN110438121A (zh) * 2018-05-03 2019-11-12 深圳华大临床检验中心 接头、接头文库及其应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015026853A2 (fr) * 2013-08-19 2015-02-26 Abbott Molecular Inc. Bibliothèques de séquençage de nouvelle génération
CN105400776A (zh) * 2014-09-12 2016-03-16 深圳华大基因科技有限公司 寡核苷酸接头及其在构建核酸测序单链环状文库中的应用
WO2016037416A1 (fr) * 2014-09-12 2016-03-17 深圳华大基因科技有限公司 Lieur vésiculaire et ses utilisations dans la construction et le séquençage d'une bibliothèque d'acides nucléiques
CN105714383A (zh) * 2014-12-22 2016-06-29 深圳华大基因研究院 一种基于分子反向探针的测序文库构建方法和试剂
CN105985945A (zh) * 2015-01-30 2016-10-05 深圳华大基因研究院 mRNA片段化方法及基于其构建测序文库的方法
WO2019149958A1 (fr) * 2018-02-05 2019-08-08 F. Hoffmann-La Roche Ag Génération de modèles d'adn circulaires à simple brin pour une molécule unique
CN110438121A (zh) * 2018-05-03 2019-11-12 深圳华大临床检验中心 接头、接头文库及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DEL PIANO ALESSIA, KECMAN TEA, SCHMID MICHAEL, BARBIERI RUGGERO, BROCCHIERI LUCIANO, TORNALETTI SILVIA, FIRRITO CLAUDIA, MINATI LU: "Phospho-RNA sequencing with circAID-p-seq", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, GB, vol. 50, no. 4, 28 February 2022 (2022-02-28), GB , pages e23 - e23, XP093118245, ISSN: 0305-1048, DOI: 10.1093/nar/gkab1158 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117845339A (zh) * 2024-01-09 2024-04-09 广西壮族自治区人民医院 一种用于检测与目标基因座相互作用的dna片段的文库构建方法
CN120060439A (zh) * 2025-02-27 2025-05-30 江苏硕世生物科技股份有限公司 一种基于封闭引物去人源核酸的测序文库构建方法及应用

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