CN117940582A - Double-end sequencing method and composition - Google Patents

Abstract

The present invention provides methods and compositions for performing nucleic acid sequencing, particularly double-ended sequencing. The method uses a multiplex sequencing template that can be generated by rolling circle amplification of an asymmetric circular nucleic acid having a central double stranded region comprising target nucleic acid sequences joined at each end to form a circular construct.

Description

Double-end sequencing method and composition

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a non-provisional utility patent application, which claims priority and benefit to the following prior provisional patent applications: USSN 63/246,188, entitled “PAIRED-END SEQUENCING METHODS AND COMPOSITIONS” filed by Jeremiah Hanes et al. on September 20, 2021, and USSN 63/219,738, entitled “PAIRED-END SEQUENCING METHODS AND COMPOSITIONS” filed by Jeremiah Hanes et al. on July 8, 2021. Each of these applications is incorporated herein by reference in its entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

not applicable.

Background technique

The development of nucleic acid sequencing technology has made countless advances in many fields. The ability to quickly and reliably determine the sequence of DNA and RNA molecules has enabled many advances in molecular biology, evolutionary biology, medical diagnostics and molecular medicine, as well as many other fields.

However, when using certain sequencing while synthesis or sequencing by combining techniques, the amount of sequence data that can be reliably obtained may be limited to a relatively small amount of bases. Although short sequence reads may be very useful in applications such as SNP analysis and genotyping, in many cases, it may be advantageous to reliably obtain further sequence data of the same template molecule. For this reason, double-ended or paired sequencing techniques have been used, for example, particularly in the context of whole genome shotgun sequencing. Double-ended sequencing can allow two "reads" of sequences to be determined from two positions of a single polynucleotide duplex. The knowledge that known double-ended sequences appear on a single duplex and are therefore connected or paired in the genome can greatly help to assemble the entire genome sequence into a consensus sequence. The additional information obtained from double-ended sequencing can also be beneficial to other applications, for example, relating to applications in which cell-free DNA is sequenced, such as the detection of circulating tumor DNA and prenatal cell-free DNA screening.

Provided herein are novel and useful compositions and methods for performing double-end sequencing. These compositions and methods provide advantages for a variety of sequencing methods, e.g., stepwise sequencing methods.

Summary of the invention

A general class of embodiments provides a method for double-end sequencing. In the method, a nucleic acid concatemer is provided, which includes multiple sequential copies of: a first adapter region, a forward strand of a target nucleic acid sequence, a second adapter region different from the first adapter region, and a reverse strand of a target nucleic acid sequence complementary to the forward strand. A sequencing process is performed to generate a double-end read of a target nucleic acid sequence by hybridizing a first sequencing primer with the first adapter region and obtaining a first read of a first portion of the target nucleic acid sequence by sequencing from the first sequencing primer, and hybridizing a second sequencing primer with the second adapter region and obtaining a second read of a second portion of the target nucleic acid sequence by sequencing from the second sequencing primer. The first read and the second read constitute a double-end read of the target nucleic acid sequence. Typically, sequencing from the first sequencing primer is completed before sequencing from the second primer, but in some embodiments, sequencing from the first primer and from the second primer is alternate or simultaneous.

In certain embodiments, nucleic acid concatemers are produced by providing circular nucleic acid molecules and using circular nucleic acid molecules as templates for rolling circle amplification to produce nucleic acid concatemers. Circular nucleic acid molecules include a central region, and the central region includes a forward strand and a complementary reverse strand of a target nucleic acid sequence. The central region, which is usually a double strand, has two ends. The forward strand utilizes a first connection region to connect the reverse strand at one end, and the forward strand utilizes a second connection region to connect the reverse strand at the other end. The first connection region and the second connection region are different from each other and are complements of the adapter region in the resulting concatemer. Circular nucleic acid molecules and nucleic acid concatemers are usually but not necessarily DNA molecules.

In one class of embodiments, rolling circle amplification is performed in solution. The resulting concatemers can then be bound to surfaces within an ordered or disordered array of other concatemers, for example, produced using the method. The position of a given concatemer in an array can be predetermined or random. Optionally, the primers extended in the rolling circle amplification reaction include a first member of a binding pair (e.g., biotin), and the surface carries a second member of a binding pair (e.g., avidin or streptavidin). In another class of embodiments, the rolling circle amplification step is performed on the surface of a solid support. For example, the primers extended in the rolling circle amplification reaction can be bound to surfaces within an ordered or disordered array of, for example, the same primers, before or after binding to the circular nucleic acid molecule and before the amplification reaction begins. Primers can be covalently or non-covalently bound to a surface (e.g., by binding pairs as described above).

In one class of embodiments, sequencing from a first sequencing primer and from a second sequencing primer is performed simultaneously. The first set of detectable signals generated by sequencing from the first sequencing primer can be distinguished from the second set of detectable signals generated by sequencing from the second sequencing primer, for example, based on their intensity. For example, by providing different concentrations of the first sequencing primer and the second sequencing primer, by providing one of the sequencing primers in the form of a mixture of extendable oligonucleotides and non-extendable oligonucleotides, and/or by using the first sequencing primer and the second sequencing primer that anneal to their respective adapter regions with significantly different efficiencies, a difference in signal intensity between the first sequencing process and the second sequencing process can be generated. Mapping with a known reference sequence can facilitate the determination of the first read and the second read.

Although in some embodiments, sequencing from the first sequencing primer and the second sequencing primer is performed alternately or simultaneously, it is more common that sequencing from the first sequencing primer is completed before sequencing from the second sequencing primer is started. In some embodiments in which sequencing from the second primer is performed after sequencing from the first primer, after obtaining the first read, the nascent chain formed by sequencing from the first sequencing primer is removed, for example, before the second sequencing primer is hybridized with the second adapter region on the concatemer. The nascent chain can be removed by any suitable process, such as cutting and washing, exonuclease digestion or denaturation. In other embodiments in which sequencing from the second primer is performed after sequencing from the first primer, the nascent chain formed by sequencing from the first primer is not removed. For example, the nascent chains can be blocked so that they do not interfere with sequencing from the second primer. Therefore, in some embodiments, the 3′ end of the nascent chain formed by sequencing from the first primer is blocked before sequencing from the second sequencing primer, usually before the second sequencing primer is hybridized with the second adapter region.

Many suitable sequencing processes are known in the art and can be applied to the practice of the inventive method. For example, sequencing from the first sequencing primer and the second sequencing primer can involve sequencing by incorporation, sequencing by connection, or sequencing by hybridization techniques. In a preferred class of embodiments, sequencing from the first sequencing primer and the second sequencing primer includes sequencing by binding techniques. During sequencing, the first sequencing primer and the second sequencing primer are optionally extended using a strand displacement polymerase. Sequencing can be performed in the presence of a single-stranded binding protein.

In some embodiments, for example, in which a polymerase lacking strand displacement activity is used, a first masked strand complementary to the forward strand is synthesized prior to sequencing from the first sequencing primer, and/or a second masked strand complementary to the reverse strand is synthesized prior to sequencing from the second sequencing primer. Typically, a first masked strand complementary to the forward strand is generated prior to hybridizing the first sequencing primer to the first adapter region, and a second masked strand complementary to the reverse strand is generated prior to hybridizing the second sequencing primer to the second adapter region. Alternatively, the first sequencing primer and the second sequencing primer can be blocked at their 3′ ends using a reversible terminator; the first primer can hybridize to the first adapter region before synthesis of the first masked strand occurs, and the second primer can hybridize to the second adapter region before synthesis of the second masked strand is performed. The reversible terminator is removed prior to initiating sequencing from the primers.

Nucleic acid concatemers generally include many copies of their repeating units, for example, at least providing a copy number of a detectable signal in the sequencing technology to be used. For example, nucleic acid concatemers may include at least 10 sequential copies of the first adapter region, the forward strand, the second adapter region, and the reverse strand, for example, at least 50, at least 100, at least 500, at least 1000, at least 5000, or at least 10,000 sequential copies. In certain embodiments, nucleic acid concatemers may include 50 to 20,000 sequential copies of the first adapter region, the forward strand, the second adapter region, and the reverse strand, for example, 50 to 10,000 or 100 to 5,000 sequential copies. It will be appreciated by those skilled in the art that the copy number used may vary, for example, depending on the type of analysis being performed. In the case where the repeating unit length is small (for example, less than several hundred bases), the copy number may be higher than the case where the repeating unit is large (for example, thousands to tens of thousands of bases). A consideration is the total molecular weight of the concatemers produced. Those skilled in the art will understand how to control the copy number and total molecular weight of the concatemers for the analysis they are performing.

Another general class of embodiments provides a method for nucleic acid sequencing. In the method, a nucleic acid concatemer is provided, which includes multiple sequential copies of: a first adapter region, a forward strand of a target nucleic acid sequence, a second adapter region different from the first adapter region, and a reverse strand of a target nucleic acid sequence complementary to the forward strand. A sequencing process is performed to determine the sequence of at least a portion of the target nucleic acid (e.g., a portion of the forward strand, a portion of the reverse strand, or both). In one class of embodiments, a masked primer is hybridized to the second adapter region and extended (e.g., using a strand displacement polymerase) to produce a first masked strand complementary to the forward strand. The first masked strand is not also complementary to the entire first adapter region. A first sequencing primer is hybridized to the first adapter region, and a first read of the first portion of the target nucleic acid sequence is obtained by sequencing from the first sequencing primer. After the first masked strand is generated, sequencing from the first sequencing primer is performed. The synthesis of the first masked strand typically but not necessarily occurs before the first sequencing primer hybridizes to the first adapter region.

By stopping the extension at the appropriate position, various strategies can be used to ensure that the first masked strand is not complementary to the entire first adapter region. In one class of embodiments, before extending the masked primer, the oligonucleotide displaced by the blocking strand is hybridized with the first adapter region. In one class of embodiments, the first adapter region includes at least one non-natural nucleotide, and the masked primer is extended under the condition of excluding the complement of the at least one non-natural nucleotide. In one class of embodiments, before the masked primer is extended, a nick is introduced into the first adapter region. For example, by hybridizing one end of a staple oligonucleotide (stapleoligonucleotide) with the first adapter region, and hybridizing the other end of the staple oligonucleotide with the second adapter region, the resulting fragment can be kept close. In certain embodiments, a single oligonucleotide can be used as both a sequencing primer and a staple oligonucleotide. Therefore, optionally, the 5' end of the first sequencing primer hybridizes with the second adapter region and/or the 5' end of the masking primer hybridizes with the first adapter region.

In cases where a double-ended read of a target nucleic acid sequence is desired, any of a variety of methods may be used to obtain a second read. The second read may be obtained from another strand of the target within the concatemer. Therefore, in one class of embodiments, after sequencing from the first sequencing primer is completed, a second masked strand is generated by further extending the nascent strand generated by sequencing from the first sequencing primer. The second masked strand is complementary to the reverse strand but not to the entire second adapter region. The first masked strand is removed. The second sequencing primer is hybridized to the second adapter region, and a second read of the second portion of the target nucleic acid sequence is obtained by sequencing from the second sequencing primer. In some embodiments, the masked primer includes a 5′ phosphate group, and the first masked strand is removed by digestion with lambda exonuclease.

The second read can be obtained from the chain generated by extending the first sequencing primer, rather than directly from the concatemer. Therefore, in one class of embodiments, the first sequencing primer includes a 5' region and a 3' region that are each hybridized to the first adapter region and are located on the flanks of the central region that is not hybridized to the first adapter region. A portion of the first adapter region remains single-stranded when the first sequencing primer hybridizes to the first adapter region. The nascent chain generated by sequencing from the first sequencing primer is further extended to produce a first extended chain complementary to the reverse chain. The first extended chain is generally not also complementary to the entire second adapter region. A displacement primer is hybridized to the single-stranded portion of the first adapter region, and a chain displacement polymerase is extended to displace the first extended chain from the reverse chain. After displacing the remainder of the extended chain, the 5' region of the first extended chain remains hybridized to the first adapter region. The second sequencing primer is hybridized to the first extended chain, and a second read of the second portion of the target nucleic acid sequence is obtained by sequencing from the second sequencing primer.

Essentially all features described above also apply in relation to these embodiments, for example with regard to the number of copies of the repeating units in the concatemers etc.

In certain embodiments, nucleic acid concatemers are produced by providing circular nucleic acid molecules and using circular nucleic acid molecules as templates for rolling circle amplification to produce nucleic acid concatemers. Circular nucleic acid molecules include a central region, and the central region includes a forward strand and a complementary reverse strand of a target nucleic acid sequence. The central region, which is usually a double strand, has two ends. The forward strand utilizes a first connection region to connect the reverse strand at one end, and the forward strand utilizes a second connection region to connect the reverse strand at the other end. The first connection region and the second connection region are different from each other and are complements of the adapter region in the resulting concatemer. Circular nucleic acid molecules and nucleic acid concatemers are usually but not necessarily DNA molecules.

In one class of embodiments, rolling circle amplification is performed in solution. The resulting concatemers can then be bound to surfaces within an ordered or disordered array of other concatemers, for example, produced using the method. The position of a given concatemer in an array can be predetermined or random. Optionally, the primers extended in the rolling circle amplification reaction include a first member of a binding pair (e.g., biotin), and the surface carries a second member of a binding pair (e.g., avidin or streptavidin). In another class of embodiments, the rolling circle amplification step is performed on the surface of a solid support. For example, the primers extended in the rolling circle amplification reaction can be bound to surfaces within an ordered or disordered array of, for example, the same primers, before or after binding to the circular nucleic acid molecule and before the amplification reaction begins. Primers can be covalently or non-covalently bound to a surface (e.g., by binding pairs as described above).

Many suitable sequencing processes are known in the art and can be applied to the practice of the inventive method. For example, sequencing from a first sequencing primer and optionally from a second sequencing primer can involve sequencing by incorporation, sequencing by ligation, or sequencing by hybridization techniques. In a preferred class of embodiments, sequencing from a first sequencing primer and optionally from a second sequencing primer includes sequencing by a binding technique. During sequencing, the first sequencing primer and the second sequencing primer are optionally extended using a polymerase lacking strand displacement activity or having a weak strand displacement activity.

Compositions, systems and kits related to, produced by or used in these methods are also features of the present invention. For example, a general class of embodiments provides a composition comprising an array of nucleic acid concatemers. The concatemers are bound to a surface, for example, with different concatemers at different sites in an orderly arrangement, or with different concatemers at different sites in a randomly distributed position in an unordered array. The position of a given concatemer in the array can be predetermined or random. Each concatemer includes multiple copies of: a first adapter region including a first sequencing primer binding site, a forward strand of a target nucleic acid sequence, a second adapter region different from the first adapter region, and a reverse strand of a target nucleic acid sequence complementary to the forward strand. The second adapter region may include a second sequencing primer binding site having a sequence different from the first sequencing primer binding site.

The concatemers can be covalently or non-covalently bound to the surface. For example, each concatemer can include a first member of a binding pair (e.g., biotin) that is bound to a second member of the binding pair (e.g., avidin or streptavidin) that in turn is bound to the surface.

In some embodiments, the first sequencing primer is hybridized to the first sequencing primer binding site. In some embodiments, the second sequencing primer is hybridized to the second sequencing primer binding site. The composition may include a nascent chain generated by extending the first sequencing primer and/or the second sequencing primer. The nascent chain generated by the extension of the first sequencing primer is optionally blocked. The composition optionally further includes: a polymerase (e.g., a strand displacement polymerase or a polymerase lacking strand displacement activity), one or more nucleotides (e.g., naturally occurring nucleotides, non-natural nucleotides, labeled nucleotides, reversible terminator nucleotides and/or chain termination nucleotides), a masked primer, a blocking oligonucleotide, a displacement primer, a masked strand, a displacement strand, one or more stapled oligonucleotides and/or other reagents used in the sequencing process. The composition is optionally present in a nucleic acid sequencing system.

Essentially all of the features described above are also relevantly applicable to these embodiments, for example, regarding the number of concatemers in the array, the number of copies of the repeating units in the concatemers, the inclusion of nucleases in the composition for removing nascent or masked strands, the inclusion of single-stranded binding proteins in the composition, suitable array substrates, etc.

Another general class of embodiments provides a kit comprising a solid support configured to bind multiple nucleic acid concatemers, a first stem-loop adapter, a second stem-loop adapter different from the first stem-loop adapter, reagents for performing rolling circle amplification (e.g., rolling circle amplification primers, strand displacement polymerases, and one or more nucleotides), a first sequencing primer, optionally a second sequencing primer, and reagents for performing nucleic acid sequencing (e.g., polymerases and one or more nucleotides, typically comprising one, two, three, or four labeled nucleotides). The polymerases used for rolling circle amplification and sequencing are typically different polymerases, but may be the same in some embodiments. The kit may also include additional reagents for generating circular nucleic acid molecules, performing rolling circle amplification to generate concatemers, and performing nucleic acid sequencing, including but not limited to a buffered reaction solution, a masking primer, a blocking oligonucleotide, a displacement primer, one or more staple oligonucleotides, a site-specific endonuclease, and/or an exonuclease. The kit also typically includes instructions for using the components, for example, for generating circular nucleic acid molecules, performing rolling circle amplification to generate concatemers, and performing nucleic acid sequencing. The components of the kit are packaged in one or more containers.

Essentially all features described above also apply to these embodiments, e.g., with respect to suitable array substrates, inclusion of single-stranded binding proteins, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 schematically illustrates an embodiment of a method for preparing nucleic acid concatemers of the present invention and performing paired-end sequencing therewith. In this embodiment, nucleic acid fragments generated in sequencing to generate a first read are removed before sequencing to generate a second read.

Figure 2 schematically illustrates an embodiment of a method for preparing a nucleic acid concatemer of the present invention and using it for double-end sequencing. In this embodiment, while sequencing is performed to generate a second read, the nucleic acid fragments generated in sequencing to generate a first read are blocked and maintained in place.

Figure 3 schematically illustrates an embodiment of a method for preparing nucleic acid concatemers of the present invention and using them for double-end sequencing. In this embodiment, a statistical mixture of symmetric and asymmetric circular nucleic acid constructs is used to generate sequenced nucleic acid concatemers.

FIG. 4 schematically illustrates the preparation of circular nucleic acid molecules and their use as rolling circle amplification templates to generate nucleic acid concatemers useful in the methods.

Figure 5 schematically illustrates an example of a method for preparing nucleic acid concatemers of the present invention and using them for nucleic acid sequencing. In this example, a masking strand is synthesized to facilitate the generation of sequence reads.

6A to 6B schematically illustrate an embodiment of a method for preparing nucleic acid concatemers of the present invention and performing paired-end sequencing therewith. In this embodiment, a masking strand is used to facilitate the generation of a first read segment and a second read segment.

7A to 7B schematically illustrate an embodiment of a method for preparing nucleic acid concatemers of the present invention and using them for double-end sequencing. In this embodiment, the nascent chain generated during the first sequencing process is extended and displaced, and a second read is obtained from the extended chain.

The schematic diagrams are not necessarily drawn to scale.

Detailed ways

Unless otherwise indicated, the practice of the present invention may use conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant technology), cell biology, biochemistry and immunology within the technical scope of the art. Such conventional techniques include polymer array synthesis, hybridization, connection, phage display and the use of markers to detect hybridization. The specific description of suitable technology can refer to the example below. However, other equivalent routine procedures can certainly be used. Such routine techniques and descriptions can be found in standard laboratory manuals, such as: Genome Analysis: A Laboratory Manual Series (Volumes I to IV), Using Antibodies: A Laboratory Manual, Cells: A Laboratory Manual, PCR Primer: A Laboratory Manual, and Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press), Stryer, L. (1995) Biochemistry (4th edition) Freeman, New York, Gait, Oligonucleotide Synthesis: A Practical Approach 1984, IRL Press, London, Nelson and Cox (2000), Lehninge Principles of Biochemistry 3rd edition, W.H. Freeman Pub., New York, NY, and Berg et al. (2002) Biochemistry 5th ed., W.H. Freeman Pub., New York, NY, all of which are incorporated herein by reference in their entirety for all purposes.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. The following definitions supplement the definitions in the art and are not attributed to any relevant or irrelevant circumstances, such as any shared patents or applications, for the current application. Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing the present invention, preferred materials and methods are described herein. Therefore, the terms used herein are only used for the purpose of describing specific embodiments and are not intended to be limited. Various other terms are defined or otherwise characterized herein.

Note that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a polymerase" refers to one reagent or a mixture of such reagents, reference to "the method" includes reference to equivalent steps and methods known to those skilled in the art, and so forth.

Where a range of values is provided, it is to be understood that each intermediate value between the upper and lower limits of the range (to one tenth of the unit of the lower limit, unless the context clearly indicates otherwise) and any other stated value or intermediate value within the stated range are encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included in smaller ranges and are also encompassed within the present invention, subject to any specifically excluded limitations in the stated ranges. Where the stated range includes one or two limits, the present invention also includes ranges excluding either or both of the included limits.

As used herein, the term "about" means that the value of a given amount varies by +/- 10% of the stated value, or optionally varies by +/- 5%, or in some embodiments, varies by +/- 1% of the stated value.

As used herein, the term "comprising" is intended to mean that compositions and methods include the stated elements, but do not exclude other elements. When used to define compositions and methods, "consisting essentially of" should mean excluding other elements that are of any importance to the compositions and methods. "Consisting of" should mean excluding other ingredients greater than trace elements and a large number of method steps of the claimed composition. Embodiments defined by each of these transitional terms are within the scope of the present invention. Therefore, it is intended that the methods and compositions may include additional steps and components (comprising), or alternatively include insignificant steps and compositions (consisting essentially of), or alternatively, only the stated method steps or compositions are intended (consisting of).

"Nucleic acid", "polynucleotide", "oligonucleotide" or grammatical equivalents herein refer to at least two nucleotides covalently linked together. The nucleic acids of the present invention generally contain phosphodiester bonds, although in some cases, nucleic acid analogs that may have alternative backbones are included, including, for example, phosphoramide, phosphorothioate, phosphorodithioate and peptide nucleic acid (PNA) backbones and bonds. Other similar nucleic acids include those with positive backbones, non-ionic backbones and non-ribose backbones, including those described in the following: U.S. Patents Nos. 5,235,033 and 5,034,506. Nucleic acids may also have other modifications, such as including heteroatoms, attaching labels (such as dyes) or replacing with functional groups that will still allow base pairing and recognition by polymerases or other enzymes. Therefore, the term "nucleic acid" encompasses any physical monomer unit string that can correspond to a nucleotide string, including nucleotide polymers (e.g., typical DNA or RNA polymers), modified oligonucleotides (e.g., oligonucleotides containing nucleotides that are not typical for biological RNA or DNA, such as 2'-O-methylated oligonucleotides), etc.

The term "single-stranded" may refer, for example, to a single polymer nucleic acid strand, or to a region within a nucleic acid polymer strand that is not base paired with a complementary region within the same strand or a different strand, depending on the context. Similarly, the term "double-stranded" may refer to two polymer strands that are hybridized to each other, or to a region of a duplex in which at least one of the participating strands also contains other portions that are not base paired, as is clear from the context.

A "nucleotide sequence" is a polymer of nucleotides (oligonucleotide, DNA, nucleic acid, etc.) or a string of characters representing a polymer of nucleotides, depending on the context. In some cases, the term "nucleotide sequence" refers to the actual sequence of bases in a nucleic acid, and in some cases, the term "nucleotide sequence" refers to a measured or determined sequence.

"Sequencing" a strand of a target nucleic acid means determining the order and identity of the nucleotides comprising the strand and/or its complement, e.g., by generating reads of the strand by any of a variety of nucleic acid sequencing techniques known in the art. Since many such techniques use primers that are complementary to the strand to be sequenced (e.g., primers that are extended as the target sequence is determined), sequencing is said to be performed "from" the primers.

A "target nucleic acid sequence" is a nucleic acid or region thereof whose nucleotide sequence or at least a portion thereof is to be determined. As is well known in the art, since specific nucleic acid sequences encode complementary sequences, any target nucleic acid sequence can be expressed as a "forward" sequence (or strand) or its complementary "reverse" sequence (or strand). Certain methods of the present invention measure a portion of the nucleotide sequence of the forward strand and a portion of the nucleotide sequence of the reverse strand (usually different portions); since the two strands are complementary, this process typically measures the nucleotide sequences of two portions of the forward strand and two portions of the reverse strand (usually two opposite ends of each). The terms "forward" and "reverse" are used herein in a relative sense rather than an absolute sense to express that one strand or sequence is the complement of another; it is not intended to imply function (e.g., coding versus non-coding), position within a chromosome, amplification sequence, or other such information.

A "read" of a nucleic acid sequence is a measured or inferred sequence of nucleotides or base pairs (or nucleotide or base pair probabilities). A read may correspond to all or part of a single DNA fragment. A read generally provides the order and identity of the measured or inferred bases in a DNA region, and may also contain other information such as quality scores, probabilities, etc.

Oligonucleotides designed to specifically anneal to fully complementary nucleic acid sequences in target nucleic acids under appropriate stringent hybridization conditions used in the present disclosure are sometimes referred to herein as "primers", and the sequences to which they anneal are referred to as "primer binding sites" (or "priming sites"). Therefore, primers having "specificity" to primer binding sites in nucleic acids (e.g., primer binding sites in adapter regions) are primers containing nucleic acid sequences that preferentially hybridize to primer binding sites under appropriate stringent hybridization conditions. Appropriate stringent hybridization conditions are generally determined by those of ordinary skill in the art. The nucleic acid sequences that hybridize to primer binding sites in primers are sometimes referred to herein as "primer regions" or similar phrases. Primers generally include regions that are not directly involved in hybridization to primer binding sites and that can have specific uses in the methods described herein. Primers can be used for different functions, including but not limited to nucleic acid synthesis and/or capturing nucleic acids containing their cognate primer binding sites. For example, a "capture primer" (or "capture oligonucleotide") can be used to isolate nucleic acids containing a specific capture primer binding site (e.g., present in an adapter or adapter region), and typically comprises a first member of a binding pair (e.g., a nucleic acid sequence, biotin, avidin, an antigen, an antibody or a binding fragment thereof, etc.) or is directly attached to a solid support. As another example, a "synthesis primer" is a primer that can be used to initiate nucleic acid synthesis by a nucleic acid polymerase (under nucleic acid synthesis conditions), and in certain specific applications, can be used as a sequencing primer in sequencing by synthesis (SBS) or sequencing by binding (SBB) applications, or as a primer in a nucleic acid amplification reaction (e.g., a rolling circle amplification reaction).

As used herein, a "substantially identical" nucleic acid is a nucleic acid having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a reference nucleic acid sequence. The length of comparison is preferably the full length of the nucleic acid, but is typically at least 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 75 nucleotides, 100 nucleotides, 125 nucleotides or more.

"Asymmetric nucleic acid" means a nucleic acid having a different nucleic acid composition at the first end of the nucleic acid compared to the second end of the nucleic acid. Asymmetric nucleic acids particularly useful in the context of the present invention are asymmetrically labeled, i.e., having a first adaptor attached to the first end and a second adaptor attached to the second end, wherein the first adaptor and the second adaptor have at least one difference in nucleic acid composition. The difference in nucleic acid composition between the first adaptor and the second adaptor can be any desired difference, including but not limited to one or more nucleic acid sequence differences (e.g., substitutions, deletions, insertions, inversions, rearrangements (e.g., different orders of functional domains), or any combination thereof). In certain embodiments, and as described in detail herein, asymmetric nucleic acids include a primer binding site for an amplification primer at one end but not at the second end.

As used herein, the term "nucleic acid concatemer" refers to a continuous nucleic acid polymer chain (e.g., a single DNA chain) containing multiple copies of the same nucleic acid sequence connected in series. Particularly useful concatemers in the context of the present invention include multiple tandem copies of the following sequences: a first adapter region, one strand of the target nucleic acid sequence, another adapter region different from the first adapter region, and a complementary strand of the target nucleic acid sequence. An "adapter region" is a nucleic acid sequence within a concatemer that connects the 3' end of a given strand of a target nucleic acid sequence to the 5' end of another strand of the target nucleic acid sequence. In embodiments of circular nucleic acids in which stem-loop adapters are connected to the ends of double-stranded fragments to form concatemers from which the concatemers are generated (e.g., by rolling circle amplification), the adapter region is complementary to the sequence provided by the adapter (i.e., each adapter region is complementary to one of the "connection regions" in the circular nucleic acid that connects the 3' end of one strand of the target nucleic acid sequence to the 5' end of another strand of the target nucleic acid sequence within the circular nucleic acid).

"Binding pair" means any two moieties that specifically bind to each other under at least one binding condition. Members of a binding pair include, but are not limited to, complementary single-stranded nucleic acid sequences, biotin/avidin or biotin/streptavidin (as well as biotin/neutravidin, biotin/traptavidin, etc.), antigen/antibody, hapten/antibody (e.g., digoxigenin/anti-digoxigenin antibody), ligand/receptor, etc. (Note that antigen/hapten binding fragments of antibodies can be used instead of whole antibodies).

"Linker" means a part that plays a role in attaching a first functional element or part to another functional element or part. With regard to attaching nucleic acid domains to each other or to different parts, linkers can be additional nucleotide residues (DNA, RNA, PNA, etc.), peptides, carbon chains, polyethylene glycol spacers, etc. The attachment of functional elements/parts to linkers can be covalent or non-covalent. There is no intention to limit in this regard.

"Strand displacement nucleic acid polymerase", "strand displacement polymerase" and their equivalents mean a nucleic acid polymerase having 5' to 3' template-dependent nucleic acid synthesis activity and 5' to 3' strand displacement activity. Therefore, when such a polymerase encounters a double-stranded region of a template during nucleic acid synthesis, it will displace the non-template strand while continuing nucleic acid synthesis on the template strand. On a circular template (e.g., a template with a double-stranded insert of hairpin adapters at both ends, as shown in the figure), such a polymerase can enter rolling circle replication under suitable nucleic acid synthesis conditions. Although any suitable strand displacement nucleic acid polymerase can be used, in certain embodiments, the polymerase is phi29 (Φ29) DNA polymerase or a modified form thereof. In the case of using a modified recombinant Φ29 DNA polymerase, it can be homologous to a wild-type or exonuclease-deficient Φ29 DNA polymerase, for example, as described in U.S. Patent Nos. 5,001,050, 5,198,543, or 5,576,204, the entire disclosure of which is incorporated herein by reference in its entirety for all purposes. Alternatively, the modified recombinant DNA polymerase can be homologous to other Φ29-type DNA polymerases, such as B103, GA-1, PZA, Φ15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, L17, For nomenclature, see also Meijer et al. (2001) "Φ29 Family of Phages". Microbiology and Molecular Biology Reviews, 65(2): 261-287. Exemplary suitable polymerases are described, for example, in U.S. Pat. Nos. 8,420,366 and 8,257,954, both entitled “Generation of modified polymerases for improved accuracy in single molecule sequencing,” U.S. Patent Application Publication Nos. 2007-0196846, 2008-0108082, 2010-0075332, 2010-0093555, 2012-0034602, 2013-0217007, 2014-0094374, and 2014-0094375, and International Patent Application Nos. WO 2007/075987, WO 2007/075873, WO 2007/075874, and WO 2007/075875. No. 2007/076057, which is incorporated herein by reference in its entirety for all purposes. Many additional suitable strand-displacing polymerases are known in the art or are commercially available.

In the following description, many specific details are set forth in order to provide a more thorough understanding of the present invention. However, it will be apparent to those skilled in the art that the present invention may be practiced without one or more of these specific details. In other cases, features and procedures well known to those skilled in the art are not described in order to avoid obscuring the present invention.

Paired-end sequencing method

The present disclosure generally relates to an improved method for nucleic acid sequencing, particularly an improved method for performing double-ended sequencing. Double-ended or paired sequencing generally relates to determining the nucleotide sequence of the first region and the second region of the target sequence of interest (e.g., determining the sequence of the two opposite ends of the nucleic acid fragment). In certain embodiments, the first region and the second region being sequenced are separated by known or unknown nucleic acid fragments that are not sequenced. In other embodiments, the first region and the second region overlap, and the sequence of the full-length target region can be determined.

In some aspects, the present invention provides for generating sequencing templates and performing double-end sequencing on these sequencing templates. The sequencing template can be a single nucleic acid chain each having the following repeating structure: adapter region 1, forward nucleic acid chain of the target, adapter region 2, reverse nucleic acid chain of the target. These nucleic acids are concatemer molecules. For example, the repeating structure can appear hundreds to thousands of times in the concatemer. The concatemer is single-stranded because it is a single polymer chain; it is obvious that under certain conditions, the chain can have a secondary structure, for example, the self-complementary parts of the chain (e.g., the forward region and the reverse region) base pair to form double-stranded regions separated by regions that remain unpaired.

Multiplexed nucleic acid sequencing templates for use in the present invention can be generated by rolling circle amplification of molecules having a central double-stranded region connected at each end by single-stranded loops to form a circular molecule. As described herein, these types of topological circular molecules are sometimes referred to as And can be produced, for example, by connecting two different hairpin (i.e., stem loop) adapters to the two ends of the double-stranded DNA fragment. The hairpin region (e.g., the region provided by the hairpin adapter) located at either end of the central self-complementary double-stranded region can be referred to as a connector region. For the present invention, the molecule preferably has a connector different from the other end at one end of the central region. In other words, the molecule is asymmetric. The molecule is asymmetric so that they form a preferred sequencing template of the present invention, wherein, for example, after the circular molecule is subjected to rolling circle amplification using an amplification primer complementary to one of the two connector regions, adapter region 1 is different from adapter region 2. Multiple nucleic acid sequencing templates can also be produced by other techniques known in the art, for example, by using multiple displacement amplification (MDA) of random or construct-specific primers. For example, the concatemer produced by rolling circle amplification of asymmetric circular nucleic acids can be used for multiple displacement amplification reactions using primers specific to the adapter-derived region of the concatemer to produce a concatemer that can be used as a sequencing template.

The present invention provides a method for sequencing a multiplex template to obtain a double-ended read of a target sequence of interest. Sequencing can be performed in any suitable manner. The sequencing method can be a step-by-step sequencing method. The sequencing method can be, for example, sequencing by synthesis (SBS), sequencing by binding (SBB) or sequencing by ligation. Since the multiplex template of the present invention comprises a clonal population of target nucleic acid sequences, the multiplex structure provides a plurality of sites for step-by-step sequencing. A single multiplex molecule has a clonal population of forward and reverse strands of a specific target region of interest. Sequencing can be performed by first introducing a sequencing primer that hybridizes with an adapter region 1. Step-by-step sequencing from the primer provides a sequence read of the first chain. The sequencing process produces a nascent nucleic acid chain complementary to the first chain. This sequencing process can be performed for a desired number of nucleotides. Typically, the read comprises a portion of the sequence of the first chain, but it can comprise the entire length of the first chain. After the first chain is subjected to such sequencing, a second sequencing primer is introduced. The second primer hybridizes with the adapter region 2, and step-by-step sequencing is performed to provide a sequencing read of a partially or fully complementary second chain. Due to the topological structure of the sequencing template, the first read corresponds to the read from the first end of the target sequence, and the second read corresponds to the read from the second end of the target sequence, thereby providing a paired end or double-ended sequence of the target sequence of interest. If the first read is one end of the "sense" strand of the target nucleic acid, the second read will be the opposite end of the "antisense" strand of the target nucleic acid (and vice versa). Base complementarity rules can be used to convert sequences or sequence regions to their complements for sequence analysis. Such analysis can use any desired sequence combination, such as the first read and the second read, the complementary sequence of the first read and the second read, or the complementary sequence of one read and another read, because all of these provide equivalent information.

When performing a second sequencing process to generate a second read, the confounding signal from the extended primer of the first sequencing process is undesirable. Therefore, in some cases, the first primer and the nascent chain generated in the first sequencing process are removed or blocked before performing the second sequencing process.

The nascent chains from the first sequencing process can be removed by any suitable process. For example, they can be degraded or they can be removed by denaturation. Degradation can be performed by enzymatic hydrolysis, such as by exonuclease treatment. In one example, the 5' end of the concatemer is protected (for example, by a biotin or bi-biotin portion, through which the concatemer is bound to the surface), and a 5' to 3' exonuclease is used to remove the nascent chains. As another example, 3' to 5' exonuclease digestion can be used to remove the nascent chains; the digestion time is usually limited so that short nascent chains are removed. Although a small part of the concatemer can also be removed during this period, the concatemer usually contains many copies of the repeating unit, so that removing a small part has little effect on the subsequent sequencing process. The nascent chains can be cut into shorter fragments that can be washed away, for example, by endonuclease treatment. Degradation can also be achieved by including nucleotides that are subsequently cleaved, for example, by incorporating uracil residues into the nascent chain during sequencing and cleaving using a mixture of uracil DNA glycosylase (UDG) and DNA glycosylase-lyase endonuclease VIII, endonuclease IV or APE1, optionally followed by washing to remove the resulting fragments. Denaturation can be performed by changing any suitable conditions such as ionic strength and/or temperature. A combination of degradation and denaturation can be used.

In some cases, before the second sequencing process is performed, the first primer and the nascent chain generated in the first sequencing process are blocked for further sequencing. There are many methods for blocking the DNA chain at the 3′ end to prevent it from generating signals that interfere with the second sequencing process. For example, a dideoxynucleotide, a 3′-blocking nucleotide (e.g., a 3′-O-azido dNTP), or another group that blocks 3′ extension can be incorporated; many such groups are known in the art. See, for example, U.S. Patent Application Publication US2020/0032322, which describes a ternary complex inhibitor portion that can be used to block the 3′ end of the nascent chain, which is incorporated herein by reference in its entirety for all purposes. The 3′ blocking group can be referred to as a terminator. In some cases, a reversible terminator can be used. For example, examples of suitable terminators are provided in the following: Chen et al. (2013) "The history and advances of reversible terminators used in new generations of sequencing technology" Journal of Genomics Proteomics Bioinformatics 11 (1): 34-40. In some cases, blocking may be irreversible. Blocking may be adjusted depending on the type of sequencing being performed. For example, if the sequencing method used in the second sequencing process is by sequencing by binding, it may be desirable to provide a blocking group that not only prevents extension, but also prevents binding of homologous nucleotides that may provide false background signals.

The method of the present invention is optionally used for highly multiplexed sequencing. Multiple sequencing templates are usually fixed to, for example, substrates. The substrate will have many multiple sequencing templates that can be distinguished individually. In some cases, optical detection of sequencing reactions is used. Other detection methods, such as electronic detection methods, can be used. The number of sequencing templates can be selected for specific applications. In some cases, millions to billions of multiple sequencing templates can be sequenced in parallel on the same substrate (for example, at least one million, at least ten million, at least one hundred million, at least one billion, at least two billion or at least three billion multiple templates). Typically, for such parallel sequencing, each multiple sequencing template will have the first adapter region and the second adapter region identical to other multiple sequencing templates, and the forward region and the reverse region in different multiple sequencing templates will represent different target molecules. For example, by fragmenting the DNA from an organism of interest, a library of such different target molecules can be produced. Many methods of forming libraries are known in the art. Because different concatemers contain the same adapter region, a pair of primers can be used to sequence different targets.

In describing various aspects of the method disclosed herein, reference will be made to the accompanying drawings. It should be understood that the accompanying drawings only illustrate specific embodiments of the disclosed method and are not intended to be limiting.

Fig. 1 schematically illustrates an embodiment of the method of the present invention. An asymmetric rolling circle amplification template 101 is provided. The circular template 101 comprises a central target region, which comprises a self-complementary forward (111) and reverse (112) region. One end of the template is covered with a first connection region 113, and the other end is covered with a second connection region 114. The first connection region 113 has a nucleic acid sequence different from that of the second connection region 114. The connection region can have sequences of various functions, such as barcodes, priming sites, restriction enzyme sites, recognition sites, etc. Note that although the connector or connection region is shown as corresponding to the single-stranded loop region of the template 101, in many cases, the connector will also have a self-complementary region extending into the double-stranded portion of the circular nucleic acid 101, for example, a hairpin adapter each of which has a single-stranded loop and a double-stranded stem is connected to a double-stranded fragment to form a circular nucleic acid. (See, e.g., the example illustrated in FIG. 4 .) One or more priming sites, barcodes, and other useful sequences (or their complements) may be included in the single-stranded or double-stranded portion (or both) of such adapters, and thus in the resulting junction region. One of the two adapters (in this example, 113) will have a rolling circle amplification initiation site, such as a rolling circle amplification primer binding site, to which primer 115 hybridizes. The amplification primer binding site is typically, but not necessarily, located in the single-stranded loop region of the junction region.

Here, in step I, the rolling circle amplification primer 115 is extended using a strand displacement polymerase such as phi29. The polymerase repeatedly travels around the circular template 101 in the direction indicated by the small arrow to form a concatemer 102 having the following repeating structure: first adapter region 123-forward strand 121-second adapter region 124-reverse strand 122 (read from 5' to 3'). The accompanying figure shows a unit of a concatemer of two copies. The arrows at the ends of the concatemer 102 indicate that there are usually more copies of the unit, such as hundreds to thousands. The number of copies can be any suitable number, such as tens, hundreds, thousands or tens of thousands of copies. Here, the first adapter region 123 is complementary to the first ligation region 113, the forward strand 121 is complementary to the reverse strand 112 in the rolling circle template 101 and is therefore substantially identical in sequence to the forward strand 111 of 101, the second adapter region 124 is complementary to the second ligation region 114, and the reverse strand 122 is complementary to the forward strand 111 in the rolling circle template 101 and is therefore substantially identical in sequence to the reverse strand 112 of 101. Although the target fragment is shown as being directly linked to a fragment complementary to the single-stranded loop region of the circular nucleic acid, it will be appreciated that there can be and typically will be other intervening sequences between these fragments. For example, where a stem-loop adapter is used to construct the template 101, the complement of one strand of the stem of the adapter will be present between the complement of the loop and the forward target region in the resulting concatemer. As described above, for rolling circle templates, these intervening sequences can be used structurally (e.g., self-complementary regions), or the intervening sequences can have specific functions, such as cleavage sites, primer binding sites, recognition sites, or barcodes comprising molecular barcodes or unique molecular identifiers (UMIs).

This rolling circle amplification (RCA) process can be carried out in solution, or can be carried out by extending the RCA primers that are bound to the substrate. In the case where the RCA primers are bound to the substrate, the product is a concatemer that is bound to the substrate by the primers. The primers can be covalently or non-covalently bound to the surface, for example, as described below or using techniques known in the art. If the RCA process is carried out in solution, the concatemers produced can be deposited and fixed to the substrate, for example, as described below or using techniques known in the art.

Step II shows a first sequencing process, which uses a first primer (131, dotted line) that hybridizes to a primer binding site in the first adapter region 123. The primer binding site on the first adapter region 123 is complementary to a sequence contained in the first connection region 113 but not present in the second connection region 114. The sequencing process can be performed on a concatemer or multiple concatemers fixed to a substrate. The sequencing process can be a step-by-step sequencing process, such as sequencing by synthesis (SBS), sequencing by binding (SBB), or sequencing by connection. In the exemplary embodiment illustrated in Figure 1, the first sequencing primer 131 extends to form a nascent chain 132, and the extension proceeds in the direction indicated by the small arrow. The sequencing process proceeds from the sequencing primer 131 to the target sequence 122 until the first sequencing process stops. This process produces a first read segment.

In this embodiment of the method, in step III, the nascent chain 132 generated by extending the primer 131 generated in the first sequencing process is removed. As described herein above, the removal can be performed by, for example, denaturation, degradation (e.g., by exonuclease), or a combination of such techniques.

In step IV, a second sequencing process is performed from a second sequencing primer 133 (bold line) that hybridizes to a primer binding site in the second adapter region 124. The primer binding site on the second adapter region 124 is complementary to a sequence contained in the second adapter 114 but not present in the first adapter 113. The second sequencing process is typically performed in the same manner as the first sequencing process, but may be performed using a different process. In the exemplary embodiment illustrated in FIG. 1 , the second sequencing primer 133 is extended to form a nascent chain 134, and the extension proceeds in the direction indicated by the small arrow. The second sequencing process is performed until the process stops. This produces a second read. The second read comes from a chain complementary to the chain from which the first read was obtained. Compared to the first read, the second read extends from the opposite end of the target nucleic acid of interest. The first read and the second read combine to provide a double-ended sequence of the target sequence of interest.

The length of the target sequence of interest will vary depending on the application. An example is a library of DNA target fragments from an organism of interest. Asymmetric hairpins are added to the ends of the target fragments to produce a library of asymmetric rolling circle amplification templates. One of the hairpins has an RCA primer binding site. The library is subjected to rolling circle amplification to form a multiple sequencing template set of the present invention. RCA can be performed in a solution, and then the multiple sequencing template set is fixed to a substrate, or RCA can extend a surface-bound primer. The multiple sequencing template set is fixed to a surface so that at least one subset of the fixed sequencing templates is individually distinguishable. The step-by-step sequencing method described herein is used to perform a first sequencing process and a second sequencing process on a sequencing template set that can be distinguished individually to produce a first read and a second read.

In some embodiments, the first read and the second read do not overlap. As an example, the target fragment can be about 500 bases long. Each of the first sequencing process and the second sequencing process can extend, for example, 150 bases. For a sequencing template with a target region length of about 500 bases, the first 150 bases and the last 150 bases of the target will be identified, while the 200 bases in the central region will not be identified. In some embodiments, for example, for a shorter target region, there may be an overlap between the first read and the second read. For example, if the target fragment is 200 bases long, and the first read and the second read are each 150 bases, then a 100 base portion will be sequenced twice in the middle of the target, thereby providing higher accuracy in the region.

The binding sites of the first sequencing primer and the second sequencing primer are usually but not necessarily located in the single-stranded loop region of their respective adapter regions. The binding site of the sequencing primer is optionally located as close as possible to the 5' end of the adapter region (e.g., as close as possible to the 5' end of the single-stranded loop portion of the adapter region) so that sequencing proceeds to the target region as early as possible in the process. Optionally, a barcode or other sequence tag is located between the 5' end of the adapter region and the sequencing primer binding site so that sequence information is generated according to the tag and the target region. One of the sequencing primer binding sites can but need not overlap with the complementary sequence of the rolling circle amplification primer binding site.

Fig. 2 schematically illustrates another embodiment of the method of the present invention. The steps are similar to the steps described in Fig. 1, but here, instead of removing the nascent chain 132 from the first sequencing process, the nascent chain 132 is blocked to provide further sequencing signals, thereby allowing the second sequencing process to be performed without removing the chain. Steps I and II are performed substantially as described above for Fig. 1. In step III, after the first sequencing process extending from primer 131 to reverse strand 122 stops, a blocker X is added, which prevents the blocked nascent chain 242 from giving an erroneous background signal during the subsequent second sequencing process. Except that the blocked nascent chain 242 remains in place during the second sequencing process, step IV is performed substantially as described above for Fig. 1. There are many known methods for blocking nascent chains in this way in the art. There are irreversible blockers, for example, they react with 3' hydroxyl to prevent further extension. Dideoxynucleotides, 3'-blocked nucleotides (e.g., 3'-O-azido dNTP) or another group that blocks 3' extension can be incorporated to terminate and block the nascent chain. Binding agents (e.g., binding proteins or antibodies) can be used to prevent extension. In some cases, sequencing polymerase can be disabled in a manner to prevent it from dissociating and stopping extension, so that it acts as a blocker. Although irreversible blockers are used in many embodiments, in other embodiments, blockers can be reversible blockers. As described above, examples of suitable reversible terminators are provided in, for example, Chen et al. (supra). For processes such as SBB, in addition to preventing extension, blockers can also be used to prevent labeled nucleotide analogs from sampling the ends of blocked nascent chains and generating background signals. This can be achieved using, for example, a binding moiety or a large-volume termination group. See, for example, U.S. Patent Application Publication US2020/0032322 (which is incorporated herein by reference in its entirety for all purposes), which describes a ternary complex inhibitor portion for blocking the 3′ end of a nascent chain, and by suppressing the formation of a ternary complex to reduce background signals in the inspection phase of sequencing by binding technology.

Typically, a sequencing process is first performed from the first primer to produce a first read, and then a second sequencing process is performed from the second primer to produce a second read. However, in some cases, it is possible to alternate between reading from the first primer and reading from the second primer. For example, different reversible blockers can be used for the first sequencing process and the second sequencing process, so that either process can be stopped and started as needed. For example, the method can be used to produce a first read and a second read, wherein each read is determined by several sequencing processes rather than a single sequencing process. If desired, the alternation between sequencing from the first primer and from the second primer can occur as frequently as each nucleotide. In other cases, sequencing from the first primer and the second primer can be performed simultaneously.

In some embodiments in which sequencing from a first sequencing primer and a second sequencing primer is performed simultaneously, signals detected during the sequencing process (e.g., optical signals observed from the binding of fluorescently labeled homologous nucleotide analogs during an SBB reaction) are distinguished based on their intensity. For example, the signal generated by sequencing from the first primer can be stronger than the signal generated by sequencing from the second sequencing primer (and vice versa). This can be accomplished, for example, by providing the first sequencing primer and the second sequencing primer at different concentrations, by providing one of the primers in the form of a mixture of extendable oligonucleotides and non-extendable oligonucleotides (thereby effectively reducing the concentration of primers that can participate in the sequencing reaction), and/or by using first and second primers that anneal to their respective adapter regions with significantly different efficiencies (e.g., by selecting primer sequences, including 2′-O-methyl nucleotides in the primers or other modifications that result in tighter or weaker primer binding, and/or by changing the size of the single-stranded loops in the adapter region, smaller loops generally reducing primer binding efficiency). Since the identification of the nucleotides in the first read and the second read is determined according to the signal observed during the sequencing process, it is possible to facilitate the determination of whether a specific signal is generated by sequencing from the first primer or the second primer (and therefore determine whether a specific nucleotide should be assigned to the first read or the second read) by mapping to a known reference sequence. For example, a possible sequence read set can be generated based on the observed signal and compared with a known reference sequence to obtain a read. In a step-by-step sequencing process, the nucleotides occupying each position can be identified in turn. In the case of simultaneous sequencing of the forward chain and the reverse chain, two nucleotides (or one nucleotide if the same nucleotide is in the next position of the two chains) are identified for each position. Therefore, for a read of length n, at most ²ⁿ possible sequences are generated for comparison with a reference. Such mapping is useful in embodiments where the intensity observed between the first sequencing process and the second sequencing process is different, and in embodiments where the intensity difference is not used and the base assignment of the first read or the second read is determined by mapping rather than by a combination of mapping and observed intensity.

Fig. 3 shows how the double-end sequencing process of the present invention is used with the statistical mixture of symmetric and asymmetric rolling circle amplification templates. In some cases, it is desirable to use a simplified process that causes the formation of symmetric and asymmetric molecules for forming asymmetric circular RCA templates. Many methods for producing such statistical mixtures are known in the art. One method is to start from a library of target DNA fragments or target DNA fragments, and a mixture of two different hairpin adapters is connected to the end. When doing so, a statistical mixture of products is produced. For example, if a hairpin adapter includes a connection region 113, and another hairpin adapter includes a connection region 114, then when two adapters of approximately equal amounts are provided, a mixture of about 50% of asymmetric circular nucleic acids 101 with both joints 113 and 114 and about 50% of symmetric circular nucleic acids (303 with two copies of the connection region 113 and 304 with two copies of the connection region 114) will be formed. Fig. 3 shows these constructs. Although using this method can reduce yield, using such statistical mixtures can be useful, for example, in the case where the process of obtaining a high percentage asymmetric construct is complicated, inconvenient, undesirable or unnecessary.

Fig. 3 shows that asymmetric rolling circle amplification template 101 works as described above. In the exemplary embodiment shown in the figure, it is shown that the nascent chain obtained from the first sequencing process is removed. Other methods described herein will also work in this context, for example, for blocking rather than removing nascent chains. In the embodiment shown in Fig. 3, the connection region 113 has a rolling circle amplification initiation site, while the connection region 114 does not. Therefore, the symmetric construct 304 containing only the connection region 114 will not undergo rolling circle amplification, and therefore will not produce a multiple sequencing template. The symmetric construct 303 has the connection region 113, and therefore has RCA initiation sites at both ends. These constructs can undergo RCA amplification to produce multiple bodies 305 as shown in step IA. Because the multiple body 305 is produced by the symmetric construct 303, it only includes the first adapter region 123. Therefore, it includes the binding site of the first sequencing primer 131 extending to the reverse region 122, and it also includes the binding site of the first sequencing primer 131 extending to the forward region 121. Therefore, when the first sequencing process is performed on the concatemer 305, there will be a convolution signal; the extension of the first sequencing primer 131 will add the base sequence to two different sequences (nascent chains 132 and 352) at the same time, so it will be difficult or even impossible to provide sequence information using the information from the concatemer without using additional information, as described below. When the second sequencing process is performed, there will be no signal from the concatemer sequencing template 305 because it does not contain the binding site of the second sequencing primer 133. Although these concatemer templates will not produce useful signals without additional operations, it is simple to identify these templates and remove them from the analysis by their characteristics (e.g., the convolution signal of the first sequencing process and/or the absence of the signal of the second sequencing process). Therefore, although the presence of symmetrical RCA templates will affect the generation of ideal concatemers and available sequence data, useful double-end sequencing data will still be obtained from those concatemer templates 102 generated by asymmetric constructs 101. However, in some cases, the convolution signal of the template such as 305 generated by the symmetrical construct can be used for sequence determination. The first read and the second read can be obtained using the mapping to a known reference sequence detailed above (eg, from a set of possible reads containing all possible combinations of one or two nucleotides identified at each position during the stepwise sequencing process).

Obviously, although in many cases it is preferred to use an asymmetric construct to achieve the double-end sequencing described in detail herein, in some cases it is not necessary to use an asymmetric construct. For example, in the case where a reference sequence is available (e.g., in a targeted sequencing method where a target or target nucleic acid sequence pool represents a specific region or region of interest), a symmetric construct can be used. In such embodiments, a symmetric construct is generated, a rolling circle amplification is performed to generate a concatemer similar to 305, and sequencing is performed from a single primer complementary to a single adapter region present in the concatemer. Then, mapping to a known reference sequence can generate a desired sequence read.

Although a symmetrical construct or a mixture of a symmetrical construct and an asymmetrical construct can be used in certain embodiments, an asymmetrical construct is preferred in many embodiments. A suitable preparation method of an asymmetrical nucleic acid used as a rolling circle amplification template in the method of the present invention is schematically illustrated in FIG. 4. In order to produce a circular nucleic acid molecule 401, two different stem-loop adapters (463 and 464) are provided and connected to the opposite ends of a double-stranded target nucleic acid fragment 466 comprising a forward strand 411 and a reverse strand 412. Therefore, an asymmetric circular nucleic acid 401 comprises a first connection region 413 (provided by adapter 463) and a second connection region 414 (provided by adapter 464) connecting the ends of a forward target strand 411 and a reverse target strand 412 to each other. In the present example, a central region 467 (which comprises the forward strand 411 and the reverse strand 412 of the target nucleic acid sequence) is initially double-stranded. Connection regions 413 and 414 each comprise a single-stranded loop and a double-stranded portion adjacent to the central target region. As detailed above, various useful sequences (or their complementary sequences) can be included in the ligation region, such as primer binding sites, barcodes or unique molecular identifiers (UMIs), restriction enzyme sites, recognition sites, etc.

In this example, a rolling circle amplification primer is hybridized to a primer binding site in the linker region 413. Primer extension by a strand displacement polymerase 465 converts the template 401 into an open circular form and generates a nucleic acid concatemer 402, as shown in FIG4. The concatemer 402 comprises multiple sequential copies of: a first adapter region 423 (which is complementary to the first linker region 413), a forward strand 421 of the target nucleic acid sequence (which is complementary to the reverse strand 412 of the circular template 401), a second adapter region 424 (which is complementary to the second linker region 414 and is therefore different from the first adapter region 423 because the linker regions 414 and 413 are different from each other) and a reverse strand 422 of the target nucleic acid sequence (which is complementary to the forward strand 411 of 401 and to the forward strand 421).

In the exemplary embodiment illustrated in FIG. 4 , the rolling circle amplification primer is hybridized to the primer binding site in the connection region 413. A portion of the first adapter region 423 (whose size depends on the position of the rolling circle amplification primer binding site in the single-stranded loop of the connection region 413), followed by the forward strand 421, thus appears at the 5′ end of the resulting concatemer. Obviously, the primer binding site can be located in the connection region 414 instead. In such embodiments, a portion of the second adapter region 424, followed by the reverse strand 422, appears at the 5′ end of the resulting concatemer. Since they are followed by the first adapter region 423, the forward strand 421, the second adapter region 424, the reverse strand 422, and so on, it is obvious that these two concatemers can be regarded as containing the same internal repeating unit. That is, no matter which connection region the rolling circle amplification primer binding site is designed into (and therefore which target strand is generated first), the resulting concatemer still contains multiple sequential copies of the following repeating units: the first adapter region, the forward strand, the second adapter region, and the reverse strand. As described above, the designation of the target strands as forward and reverse is purely for convenience in referring to the two complementary strands. There is therefore no restriction on which strand is amplified first. The designation of the adapter regions, linker regions, etc. as first and second is also purely for convenience in referring to these elements.

Concatemers can, for example, be fixed on the surface at distinguishable points or positions in the disordered distribution of ordered arrays or such concatemers, for example, fixed on the surface of a slide, a chip, a flow cell or other suitable substrates. The position of a given concatemer in an ordered or disordered array can be predetermined or random. Concatemers can be fixed on, for example, a flat surface of a substrate, a non-flat surface of a substrate or fixed in a substrate in a three-dimensional manner, such as in a gel matrix. Fixation on substrates can be carried out in any suitable manner. In certain embodiments, concatemers are produced on the surface. For example, rolling circle amplification primers can be fixed on the surface, for example, covalently, by combining the biotin or double biotin groups on the primers via avidin or streptavidin with the biotin or double biotin of the binding surface, by hybridizing with complementary oligonucleotides fixed on the surface or by arrays known in the art for producing oligonucleotides or otherwise fixing any other technology of oligonucleotides. (See, e.g., U.S. Pat. No. 6,274,320, which describes techniques for producing an array of attachment sites on a solid support and attaching primers thereto.) Primer extension then results in the fixation of the concatemer at that location. In other embodiments, the concatemer is produced in solution and then fixed by any technique known in the art for producing an array of nucleic acids or otherwise fixing nucleic acids. For example, a biotinylated rolling circle amplification primer can be extended in solution, and the resulting biotinylated concatemer can then be fixed on a biotinylated surface by avidin or streptavidin. As another example, the concatemer can be fixed by hybridizing with one or more oligonucleotides bound to a surface. As yet another example, the concatemer can be fixed by electrostatic interactions with a positively charged surface. As yet another example, the concatemer can be covalently attached to a surface, e.g., by one or more functional groups contained in a rolling circle amplification primer and/or the concatemer itself; for example, a click chemistry group can be included in one or more nucleotides in a primer and/or incorporated into one or more nucleotides in a concatemer during rolling circle amplification. See, e.g., U.S. Patent Application Serial No. 17/575,094, which describes exemplary suitable surfaces and concatemers attached thereto, and which is incorporated herein by reference in its entirety for all purposes. In the case where a concatemer population is fixed on a surface, their positions can be preselected so that they are at a sufficient distance apart so that the signal generated for a given concatemer during the sequencing process can be distinguished from the signal generated by other concatemers. Similarly, in the case where a concatemer population is fixed at a randomly determined position on a surface, their density can be controlled so that at least some concatemers are at a sufficient distance apart so that the signal generated for a given concatemer during the sequencing process can be distinguished from the signal generated by other concatemers (e.g., by diluting the concatemers before deposition and/or by limiting the density of available attachment sites on the surface).

Under certain conditions, the forward strand and reverse strand in the concatemer will hybridize with each other when they are produced, so that the concatemer contains double-stranded regions separated by single-stranded regions complementary to the single-stranded loop in the rolling circle amplification template. Therefore, it may be advantageous to use a strand displacement polymerase in the first sequencing process and the second sequencing process. (This polymerase may be the same or different from any strand displacement polymerase used in the aforementioned rolling circle amplification reaction.) It is also possible to use a technique to reduce the formation of secondary structures by preventing or eliminating the hybridization of the forward strand and the reverse strand in the concatemer, instead of using a strand displacement polymerase or in combination with a strand displacement polymerase. For example, a single-stranded DNA binding protein (e.g., an Escherichia coli single-stranded DNA binding protein) can be provided in an amount sufficient to maintain the forward strand and the reverse strand in a single-stranded form during rolling circle amplification and/or sequencing processes. Various suitable single-stranded binding proteins (SSBs) are known in the art, and purification techniques thereof are also known. In addition, single-stranded DNA binding proteins, such as single-stranded DNA binding proteins from Escherichia coli, are commercially available from suppliers such as Thermo Fisher Scientific and AS One International.

As another example, a masked strand complementary to one of the target strands can be generated to ensure that the other strand is in a single-stranded form, so that it is easy to sequence with a polymerase that lacks or has a weak strand displacement activity. Figure 5 schematically illustrates an embodiment of the method of the present invention, in which a masked strand is used to facilitate sequencing using a polymerase that lacks a strong strand displacement activity. An asymmetric circular rolling circle amplification template 501 is provided. The circular template 501 includes a central target region, which includes a self-complementary forward (511) and reverse (512) region. One end of the template is covered with a first connection region 513, and the other end is covered with a second connection region 514. The first connection region 513 has a nucleic acid sequence different from that of the second connection region 514. The connection region can have sequences of various functions, such as barcodes, priming sites, restriction enzyme sites, recognition sites, etc. Note that although the joint or connection region is shown as a single-stranded loop region corresponding to template 501, in many cases, the joint will also have a self-complementary region extending into the double-stranded portion of the circular nucleic acid 501, for example, each of which has a hairpin adapter with a single-stranded loop and a double-stranded stem is connected to a double-stranded fragment to form a circular nucleic acid. (See, for example, the example illustrated in Fig. 4.) One or more priming sites, barcodes and other useful sequences (or their complementary sequences) can be included in the single-stranded or double-stranded portion (or both) of such adapters, and are therefore included in the resulting connection region. One of the two joints (513 in this example) has a rolling circle amplification start site that primer 515 hybridizes with it, such as a rolling circle amplification primer binding site. The amplification primer binding site is usually but not necessarily located in the single-stranded loop region of the connection region.

Here, in step I, the rolling circle amplification primer 515 is extended using a strand displacement polymerase such as phi29. The polymerase repeatedly moves around the circular template 501 in the direction indicated by the small arrow to form a concatemer 502 having the following repeating structure: the first adapter region 523-forward chain 521-second adapter region 524-reverse chain 522. Only a small part of the middle of the concatemer is shown in the figure; the arrows at both ends of the concatemer 502 indicate that there are usually many copies of the repeating unit, such as hundreds to thousands. The number of copies can be any suitable number, such as tens, hundreds, thousands or tens of thousands of copies. Here, the first adapter region 523 is complementary to the first connection region 513, the forward chain 521 is complementary to the reverse chain 512 in the rolling circle template 501 and the sequence is therefore substantially the same as the forward chain 511 of 501, the second adapter region 524 is complementary to the second connection region 514, and the reverse chain 522 is complementary to the forward chain 511 in the rolling circle template 501 and the sequence is therefore substantially the same as the reverse chain 512 of 501. Although the target fragment is shown as being directly connected to the fragment complementary to the single-stranded loop region of the circular nucleic acid, it is understood that there can be and typically will be other intervening sequences between these fragments. For example, where a stem-loop adapter is used to construct template 501, the complement of one strand of the stem of the adapter will be present between the complement of the loop and the forward target region in the resulting concatemer. As described above, for rolling circle templates, these intervening sequences (e.g., self-complementary regions) can be used structurally, or the intervening sequences can have specific functions, such as cleavage sites, primer binding sites, recognition sites, or barcodes comprising molecular barcodes or unique molecular identifiers (UMIs).

This rolling circle amplification (RCA) process can be carried out in solution, or can be carried out by extending the RCA primers bound to the substrate. In the case where the RCA primers are bound to the substrate, the product is a concatemer bound to the substrate by the primers. The primers can be covalently or non-covalently bound to the surface, for example, as described herein or using techniques known in the art. If the RCA process is carried out in solution, the concatemers produced can be deposited and fixed on the substrate, for example, as described herein or using techniques known in the art.

In step II, masked primer 575 hybridizes with a binding site in second adapter region 524. The primer binding site on second adapter region 524 is complementary to a sequence contained in second connection region 514 but not in first connection region 513. In step III, masked primer 575 is typically extended by a strand displacement polymerase to form a first masked strand 576 complementary to forward strand 521. The extension of masked primer 575 stops before it travels through the entire first adapter region 523, so that the resulting first masked strand 576 is not complementary to the entire first adapter region 523. In the scheme illustrated in FIG5 , extension is stopped at a position indicated by X in the first adapter region 523. Any of a variety of methods can be used to block extension, and they can be combined if necessary. Exemplary methods are described in detail below. In addition, for any of these exemplary methods, extension can optionally be performed at low temperatures and/or a polymerase with relatively weak strand displacement activity can be used to facilitate stopping extension at a desired point.

In an exemplary method, extension can be stopped by hybridizing an oligonucleotide blocking strand displacement to a single-stranded loop region of the first adapter region. Exemplary oligonucleotides capable of blocking strand displacement by strong hybridization with its binding site include, for example, oligomers comprising locked nucleic acid (LNA) and/or peptide nucleic acid (PNA) residues. Other exemplary blocking oligonucleotides can form one or more interchain crosslinks with the first adapter region. For example, a blocking oligonucleotide can include at least one 5-bromo-deoxyuridine (available from, for example, Integrated DNA Technologies, Inc.), which forms a crosslink with the first adapter region when exposed to ultraviolet light.

As another example, extension can be stopped by including at least one non-natural nucleotide in the first adapter region and extending the masked primer under conditions that exclude the complement of the at least one non-natural nucleotide. A variety of non-natural bases that cannot effectively base pair with natural bases are known in the art and can be used in the practice of the present invention. For example, one or more nucleotide residues containing isocytosine (isoC) can be included in the loop region of one of the stem-loop adapters used to prepare the rolling circle amplification template, so that the first connection region of the resulting rolling circle amplification template contains one or more isoC. The rolling circle amplification primer is extended in a mixture containing nucleotides including isoguanine (isoG) so that the resulting concatemer contains one or more isoG. When the masked primer is extended, isoC is not provided in the reaction mixture, so the extension cannot proceed through the template position occupied by isoG.

As another example, before extending the masking primer, a nick can be introduced into the first adapter region to stop extension at the nick. The masking primer can be nicked after hybridization with the concatemer or more usually before it. The nick can be introduced, for example, using an endonuclease with a specific recognition site. A variety of suitable site-specific endonucleases are known in the art and can be commercially available, for example, from New England Biotechnology Company (New England Biosciences, Inc.). As an example, the recognition site of the nicking endonuclease can be designed in the double-stranded stem portion of the first adapter region, or the oligonucleotide can be hybridized with the single-stranded loop portion of the first adapter region to produce the recognition site of the nicking endonuclease. Only a restriction endonuclease or a naturally occurring nicking endonuclease that hydrolyzes only one chain of the duplex can be used. As another example, the oligonucleotide can be hybridized with the single-stranded loop portion of the first adapter region to produce a recognition site for the endonuclease that cuts two chains (cutting the oligonucleotide and leaving a nick in the adapter region). For example, a methylated oligonucleotide complementary to the single-stranded portion of the first adaptor region can create a site for cleavage by FspEI (New England Biotech). As yet another example, Tth Argonaute (New England Biotech) can be used with a 5′-phosphorylated single-stranded DNA guide to introduce a nick in the portion of the adaptor region having a strand complementary to the guide.

In some embodiments, for example, in the case of sequencing using an SBB technique in which a signal from a fluorescently labeled homologous nucleotide bound to the next available base in the template is detected to identify the next correct nucleotide in the sequence (particularly in the case of sequencing multiple targets simultaneously at different locations on the surface of a flow cell or other substrate), bringing the fragments produced by the nicked concatemers close to each other can increase the signal intensity observable from the cluster of fragments (i.e., for the specific target nucleic acid). The fragments produced by the nicked concatemers are optionally held together by hybridizing one or more staple oligonucleotides similar to those used in DNA origami to the fragments. Such hybridization can bridge different fragments, allowing the fragments produced by a given concatemer to be tightly aggregated.

Each staple oligonucleotide can be combined with two or more fragments (directly or indirectly) at the same time to keep them close to each other. In various exemplary designs, the staple oligonucleotide can be directly hybridized with the adapter region of two or more fragments, can be hybridized with a fragment and another staple oligonucleotide, and/or can be hybridized with a fragment and combined with an intermediate. For example, a portion of a staple oligonucleotide (e.g., one end of a staple oligonucleotide) can be hybridized with a single-stranded portion of a first adapter region, while another portion of a staple oligonucleotide (e.g., its other end) can be hybridized with a single-stranded portion of a second adapter region, such as on different fragments. As another example, a portion of a staple oligonucleotide (e.g., one end of a staple oligonucleotide) can be hybridized with a single-stranded portion of a first adapter region, while another portion of a staple oligonucleotide (e.g., its other end) can be hybridized with a single-stranded portion of a first adapter region in another case; the portion of the first adapter region that is hybridized with a staple oligonucleotide optionally has the same sequence. Similarly, a staple oligonucleotide can be hybridized with a single-stranded portion of a second adapter region in two different cases. In some aspects, the parts of staple oligonucleotides can be mutually hybridized or hybridized with intermediates.For example, the parts of two or more staple oligonucleotides can be hybridized with the same single intermediate oligonucleotide or different intermediate oligonucleotides, such as intermediate oligonucleotides presented by particles.The staple oligonucleotides can be functionalized (for example, at its 3' end and/or 5' end and/or inside) so as to be cross-linked to each other or by intermediates.For example, biotinylated staple oligonucleotides can be cross-linked by streptavidin (for example, by adding streptavidin after the staple oligonucleotide is hybridized with the first adapter region and/or the second adapter region).In another example, staple oligonucleotides can be functionalized using click chemistry groups such as strain-promoted click chemistry groups, and can be cross-linked by presenting the multivalent intermediates of complementary click chemistry partners in a variety of situations.Exemplary strain-promoted click chemistry groups and partners include but are not limited to dibenzocyclooctene (DBCO) and azide, trans-cyclooctene (TCO) and tetrazine, and derivatives thereof. For example, DBCO-functionalized stapled oligonucleotides can be cross-linked via dendrimers presenting multiple azides.

A single oligonucleotide may optionally be used as a primer and a staple oligonucleotide. For example, the 5' end of the first sequencing primer may hybridize to the second adapter region, while the 3' end of the first sequencing primer hybridizes to the first adapter region (so that the first sequencing primer also serves as a staple oligonucleotide), and/or the 5' end of the masking primer may hybridize to the first adapter region, while the 3' end of the masking primer hybridizes to the second adapter region (so that the masking primer also serves as a staple oligonucleotide). Obviously, the portions of the adapter regions that are complementary to the sequencing, masking, and staple portions of such primers will generally not overlap with each other, so that the primers will not compete for their binding sites.

In the example illustrated in FIG5 , after synthesizing the first masked strand 576, in step IV, the first sequencing primer 531 hybridizes with the primer binding site in the first adapter region 523. The primer binding site on the first adapter region 523 is complementary to a sequence contained in the first connection region 513 but not present in the second connection region 514. The sequencing process can be performed on a concatemer or multiple concatemers (e.g., thousands to millions to billions) fixed on a substrate. The sequencing process can be a step-by-step sequencing process, such as SBS, SBB, or sequencing by connection. In the exemplary embodiment illustrated in FIG5 , the first sequencing primer 531 is extended in step V to form a nascent chain 532, wherein the extension proceeds in the direction indicated by the arrow. The sequencing process proceeds from the sequencing primer 531 to the reverse strand 522 of the target sequence until the first sequencing process stops. This process produces a first read segment.

Where paired-end reads of a target nucleic acid sequence are desired, any of a variety of methods may be used to obtain the second read. Two exemplary methods are described in detail below.

Figures 6A to 6B schematically illustrate an embodiment in which a second read is obtained from another strand of the target within the concatemer. In this embodiment, the masking primer 675 includes a 5' phosphate group, and therefore the first masked strand 676 also includes a 5' phosphate group. In other aspects, steps I to V are performed as detailed above with respect to Figure 5. After the sequencing from the first sequencing primer 531 is completed, the nascent strand 532 generated by sequencing from the first sequencing primer 531 is extended in step VI to generate a second masked strand 638. The extension of the nascent strand 532 is stopped before it travels through the entire second adapter region 524, so that the resulting second masked strand 638 is not complementary to the entire second adapter region 524. The first sequencing primer 531 may be extended using a polymerase lacking strand displacement activity such that extension stops at the 5' end of the first masked strand 676, or the masked primer 675 and thus the first masked strand 676 may comprise LNA or PNA residues (or other modifications that result in tight binding to the concatemer), interstrand crosslinks may be formed between the masked primer 675 and the second adapter region 524, or the second adapter region 524 may comprise at least one non-natural nucleotide, the complement of which is not included in the extension reaction, so as to stop extension as described in detail above. After the second masked strand 638 is synthesized, the first masked strand 676 is removed in step VII, for example, by digestion with lambda exonuclease or a similar enzyme that catalyzes the removal of 5' to 3' nucleotides from double-stranded DNA having a 5' phosphate.

In step VIII, the second sequencing primer 633 hybridizes with the primer binding site in the second adapter region 524. The primer binding site on the second adapter region 524 is complementary to a sequence contained in the second adapter 514 but not present in the first adapter 513. In step IX, a second sequencing process is performed from the second sequencing primer 633. The second sequencing process is usually performed in the same manner as the first sequencing process, but can be performed using different processes. In the exemplary embodiment illustrated in Figures 6A to 6B, the second sequencing primer 633 is extended to form a nascent chain 634, wherein the extension proceeds in the direction indicated by the arrow. The second sequencing process is performed until the process stops. This produces a second read. The second read comes from a chain complementary to the chain from which the first read is obtained. Compared to the first read, the second read extends from the opposite end of the target nucleic acid of interest. The first read and the second read combine to provide a double-ended sequence of the target sequence of interest.

7A-7B schematically illustrate an embodiment in which the second read is obtained from a strand formed by extending the first sequencing primer rather than directly from a concatemer. In this embodiment, the masked primer 775 and therefore also the first masked strand 776 comprises LNA residues, PNA residues, or other modifications that result in strong binding to the second adapter region 524. In other aspects, steps I to III are performed as detailed above for FIG. 5 .

In the example illustrated in FIGS. 7A to 7B , after synthesizing the first masked strand 776, in step IV, the first sequencing primer 731 is hybridized to the first adapter region 523. The first sequencing primer 731 includes a region located at or near its 5′ end and a region located at or near its 3′ end, each of which hybridizes to the first adapter region 523. These 5′ and 3′ regions of the first sequencing primer 731 are located on the flanks of the central region that is not complementary to and does not hybridize with the first adapter region 523. The primer binding subsites on the first adapter region 523 are separated by regions that are not complementary to the first sequencing primer 731, so that a portion of the first adapter region 523 remains single-stranded. In the exemplary embodiment illustrated in FIGS. 7A to 7B , the first sequencing primer 731 is extended in step V to form a nascent strand 732, wherein the extension proceeds in the direction indicated by the arrow. The sequencing process can be a stepwise sequencing process, such as SBS, SBB, or sequencing by ligation, and can be performed on one or more concatemers fixed on a substrate. The sequencing process proceeds from the first sequencing primer 731 to the reverse strand 522 of the target sequence until the first sequencing process stops. This process generates a first read segment.

As illustrated in Figures 7A-7B, in step VI, the nascent strand 732 generated by sequencing from the first sequencing primer 731 is further extended to generate an extended strand 782 that is complementary to the reverse strand 522. When the tightly bound 5' end of the first masked strand 776 is reached, the extension stops, so that the extended strand 782 is not complementary to the entire second adapter region 524 (although it is typically complementary to a portion of the second adapter region to provide a convenient universal binding site for the second sequencing primer 733). Obviously, in addition to or instead of including LNA in the first masked primer 775 and the first masked strand 776, other techniques for terminating extension (e.g., nicking the second adapter region 524 or including non-natural nucleotides in the second linker region 524 and excluding their complementary nucleotides from the extension reaction) may also be used.

In step VII, the displacement primer 785 hybridizes to the single-stranded portion of the first adapter region 523 between the regions that bind the first sequencing primer 731. In step VIII, the second masked strand 786 is generated when the displacement primer 785 is extended, typically by a strand displacement polymerase that displaces the extended strand 782 from the reverse strand 522. The 5′ region of the extended strand 782 remains hybridized to the first adapter region 523. In the embodiment illustrated in FIGS. 7A-7B , extension of the displacement primer 785 stops when the tightly bound 5′ end of the first masked strand 776 is reached. Likewise, it will be apparent that other techniques for terminating extension (e.g., nicking the second adapter region 524 or including non-natural nucleotides in the second linker region 524 and excluding their complementary nucleotides from the extension reaction) described in detail above may be used in addition to or in lieu of including LNAs in the first masked primer 775 and the first masked strand 776. In some embodiments, extension is allowed to proceed through the second linker region 524; in such embodiments, extension can be terminated in the first linker region 523 instead using the techniques detailed above (e.g., nicking the first linker region 523 or including non-natural nucleotides in the first linker region 523 and excluding their complementary nucleotides from the extension reaction).

In step IX, the second sequencing primer 733 hybridizes with the primer binding site in the displaced extended strand 782. In step X, a second sequencing process is performed from the second sequencing primer 733. The second sequencing process is usually performed in the same manner as the first sequencing process, but can be performed using different processes. In the exemplary embodiment illustrated in Figures 7A to 7B, the second sequencing primer 733 is extended to form a nascent strand 734, wherein the extension proceeds in the direction indicated by the arrow. The second sequencing process is performed until the process stops. This produces a second read. The second read comes from a strand complementary to the strand from which the first read is obtained. Compared to the first read, the second read extends from the opposite end of the target nucleic acid of interest. The first read and the second read combine to provide a double-ended sequence of the target sequence of interest.

Although the above examples have been described for paired-end sequencing, it is clear that each of these methods can be used to determine the nucleic acid sequence of a single region of a target by performing only those steps that result in sequencing from a first sequencing primer to obtain a first read, without continuing with the steps that result in sequencing from a second sequencing primer to obtain a second read. Such methods are also a feature of the present invention.

Generating rolling circle amplification template

Typically, the nucleic acid rolling circle amplification template for specific use in the context of the present invention comprises a double-stranded region with a hairpin at each end (e.g., a double-stranded nucleic acid insert containing a target nucleic acid sequence of interest). Such constructs can be generated using any suitable method. In certain embodiments, as known in the art, hairpin ends are attached to double-stranded nucleic acid fragments by connecting hairpin adapters to the compatible ends of the fragments, for example, via blunt ends or sticky ends. The connection of hairpin adapters produces a nucleic acid construct comprising a single-stranded region, which connects two chains of double-stranded nucleic acid inserts (e.g., by the stem of a hairpin adapter, the 3' end of the first chain of the insert is connected to the 5' end of the hybridization complementary chain of the insert, the 3' end of the adapter is connected to the 5' end of one chain of the insert, and the 5' end of the adapter is connected to the 3' end of another chain of the insert, as illustrated in Figure 4). As required, single-stranded regions can be used for initiation and/or capture. Other methods for generating hairpin ends on nucleic acids can be used.

Circular nucleic acids found to be useful in the present disclosure include A template is a nucleic acid having a central double-stranded region and a hairpin region at each end of the double-stranded region. A circular template such as Preparation and use of templates are described, for example, in U.S. Pat. No. 8,153,375, U.S. Pat. No. 8,236,499, and Travers et al. (2010) Nucl. Acids Res. 38(15):e159, the entire disclosures of which are hereby incorporated by reference for all purposes. /> One advantage of the template is that it can be made from a library of double-stranded nucleic acid (e.g., DNA) fragments. For example, a genomic DNA sample can be fragmented into a library of DNA fragments by known methods, such as by shearing or by using restriction endonucleases. The library of DNA fragments can be ligated to hairpins or other stem-loop adapters at each end of the fragments to produce a library of double-stranded nucleic acid (e.g., DNA) fragments. The hairpin adapters provide a single-stranded region within the hairpin, which provides a useful location for the complement of the rolling circle amplification primer binding site and the sequencing primer binding site. By using the same hairpin adapter pair for all fragments, the hairpin adapters provide a location for universal priming of all target sequences.

As described above with respect to FIG. 3, a mixture of two different hairpin adapters is connected to a double-stranded target fragment to obtain a mixture of symmetrical circular nucleic acids (having the same adapter at both ends) and asymmetric circular nucleic acids (having different adapters at both ends). Although the mixture can be used for sequencing of the target as described in detail above, the asymmetric circular construct can also be purified from the mixture, for example, as described in detail in U.S. Patent No. 10,920,268, which is hereby incorporated by reference as a whole for all purposes. For example, a symmetrical circular nucleic acid comprising two copies of the first hairpin adapter and an asymmetric circular nucleic acid comprising the first hairpin adapter and the second hairpin adapter can be captured on a bead carrying a capture primer complementary to the first adapter. Symmetric circular nucleic acids with two copies of the second adapter are not captured and are therefore removed from the mixture. Then a primer complementary to the second adapter can be extended using a strand displacement polymerase, and this extension of the primer elutes the asymmetric nucleic acid from the bead. Other techniques for purifying asymmetric circular nucleic acids are also described in detail in U.S. Patent No. 10,920,268.

Obviously, in addition to purifying asymmetric nucleic acids from a mixture of symmetrical and asymmetric constructs or replacing the purification of asymmetric nucleic acids from a mixture, the desired concatemers can also be purified from a mixture of concatemers. For example, a mixture of a first hairpin adapter including a rolling circle amplification primer binding site and a second hairpin adapter lacking a rolling circle amplification primer binding site can be connected to a double-stranded target fragment to obtain a mixture of symmetrical circular nucleic acids (having the same adapter at both ends) and asymmetric circular nucleic acids (having different adapters at both ends). When rolling circle amplification is performed, only asymmetric constructs and those symmetrical constructs with two first adapters will be amplified; symmetrical constructs with two second adapters cannot hybridize with primers and therefore will not be amplified. By binding an oligonucleotide fixed on a solid support to a binding site complementary to the second adapter in the concatemer region, the resulting concatemer can be exposed to a solid support (e.g., a bead, the surface of a flow cell or other sequencing substrate, etc.). Therefore, only the concatemers generated by the amplification of the asymmetric construct will be captured. These concatemers can be used as sequencing templates as detailed herein, or they can be subjected to multiple displacement amplification and the resulting concatemer products can be used as sequencing templates as detailed herein.

The technology of constructing asymmetric nucleic acid colony can be used, rather than purifying asymmetric nucleic acid from the mixture of symmetric construct and asymmetric construct. Exemplary suitable methods for generating asymmetric circular nucleic acid are described in, for example, U.S. Patent No. 10,370,701, which is hereby incorporated by reference as a whole for all purposes. For example, a symmetrical circular nucleic acid including a nick is constructed between a hairpin adapter and the double-stranded insert at each end. Extend from the nick, and different hairpin adapters are connected to the free end of the resulting product. Other methods for generating a nucleic acid construct suitable for use as asymmetric rolling circle amplification template of the present invention are provided in, for example, U.S. Patent Application Publication No. 2012/0196279, which is hereby incorporated by reference as a whole for all purposes.

Some useful methods for generating rolling circle amplification templates used in the methods of the invention start with double-stranded nucleic acid fragments with defined ends, which can be blunt ends or ends with known overhang sequences (5' or 3' overhang sequences). These nucleic acid fragments can be of any size or size range and can comprise DNA, RNA, DNA-RNA hybrids (e.g., molecules having one mRNA strand and one complementary DNA strand produced by synthesizing the first strand during the preparation of cDNA), genomic DNA, cDNA, mRNA, tRNA, etc. In some embodiments, the nucleotide sequence of the fragment is unknown.

In certain embodiments, the double-stranded nucleic acid fragments used in the methods and compositions of the present disclosure include nucleic acids obtained from samples. The sample may contain any number of substances, including but not limited to: body fluids (including but not limited to blood, urine, serum, lymph, saliva, anal and vaginal secretions, sweat and semen) and cells of almost any organism (e.g., mammalian species including humans); environmental samples (including but not limited to air, agricultural, water and soil samples); biological warfare agent samples; research samples; products of amplification reactions (including both target and signal amplification, such as PCR amplification reactions); purified samples (e.g., such as purified genomic DNA, original samples (bacteria, viruses, genomic DNA, etc.)). As will be appreciated by those skilled in the art, almost any experimental operation may be performed on the sample.

When used in the disclosed methods, genomic DNA can be prepared from essentially any source by three steps: cell lysis, deproteinization, and DNA recovery. These steps are adapted to the requirements of the application, the required yield, purity, and molecular weight of the DNA, and the amount and history of the source. Further details on genomic DNA isolation can be found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, Inc., San Diego, CA (Berger); Sambrook et al., Molecular Cloning—A Laboratory Manual (3rd ed.), Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 2008 (“Sambrook”); Current Protocols in Molecular Biology, FM Ausubel et al., eds., Current Protocols, a joint venture of Green Publishing Associates and John Wiley & Sons, Inc. (supplements through 2021) (“Ausubel”); Kaufman et al. (2003) Handbook of Molecular and Cellular Methods in Biology and Medicine Second Edition. Edition) Ceske (ed.) CRC Press (Kaufman); and The Nucleic Acid Protocols Handbook Ralph Rapley (ed.) (2000) Cold Spring Harbor Laboratory, Humana Press Inc (Rapley). In addition, a number of commercially available kits are used to purify genomic DNA from cells, including: Wizard ^™ Genomic DNA Purification Kit, available from Promega; Aqua Pure ^™ Genomic DNA Isolation Kit, available from BioRad; Easy-DNA ^™ Kit, available from Thermo Fisher Scientific; and DnEasy ^™ Tissue Kit, available from Qiagen, Germany. Alternatively or additionally, target nucleic acid fragments can be obtained by a targeted capture protocol, for example, where the target nucleic acid is initially obtained as a single-stranded fragment on a microarray or other capture technology, and the captured material is subsequently amplified to generate double-stranded sample material. A variety of such capture schemes have been described, for example, in: Hodges E et al., Nat. Genet. 2007 Nov 4, Olson M., Nature Methods 2007 Nov;4(11):891-2, Albert TJ et al., Nature Methods 2007 Nov;4(11):903-5, and Okou DT et al., Nature Methods 2007 Nov;4(11):907-9.

The nucleic acid that can be used in the methods described herein can also be derived from cDNA, such as cDNA prepared from mRNA obtained from, for example, a eukaryotic subject or a specific tissue derived from a eukaryotic subject. For example, the data obtained by sequencing a nucleic acid target derived from a cDNA library using a high-throughput sequencing system can be used to identify, for example, new splicing variants of a gene of interest, or to compare, for example, differential expression of splicing isoforms of a gene of interest between, for example, different tissue types, between different treatments of the same tissue type, or between different developmental stages of the same tissue type.

Generally, the scheme and method described in Sambrook and Ausubel can be used to separate mRNA from almost any source. The output and quality of the mRNA separated can depend on how to store tissue, extract the means of destroying tissue during RNA extraction, or extract the tissue type of RNA therefrom before RNA extraction. RNA separation scheme can be optimized accordingly. Many mRNA separation kits can be commercially available from Thermo Fisher Scientific and Technological Company and U.S. Bo Cheng Company (BioChain). In addition, mRNA from various sources (for example, cattle, mice and people) and tissue (for example brain, blood and heart) can be commercially available from U.S. Bo Cheng Company (Hayward, CA, CA) and Takara Bio Engineering Company (Takara Bio) (San Jose, CA, San Jose, CA).

Once the purified mRNA is recovered, reverse transcriptase is used to generate cDNA from the mRNA template. Methods and protocols for generating cDNA from mRNA (e.g., mRNA harvested from prokaryotes and eukaryotes) are described in cDNA Library Protocols, IG Cowell et al., eds., Humana Press, New Jersey, 1997, Sambrook and Ausubel. In addition, many kits are commercially available for preparing cDNA, including Cells-to-cDNA ^™ II, RETROscript ^™ , and CloneMiner ^™ cDNA Library Construction Kits (Thermo Fisher Scientific) and Universal cDNA Synthesis System (Promega). Many companies, such as Creative Biogene and GenScript, provide cDNA synthesis services.

In some embodiments of the invention described herein, nucleic acid fragments are generated from genomic DNA or cDNA. There are many methods for generating nucleic acid fragments from genomic DNA, cDNA or DNA concatemers. These methods include, but are not limited to: mechanical methods, such as ultrasonic treatment, mechanical shearing, atomization, hydraulic shearing, etc.; chemical methods, such as treatment with hydroxyl radicals, Cu(II): thiol combinations, diazonium salts, etc.; enzymatic methods, such as exonuclease digestion, restriction endonuclease digestion, transposon cleavage and labeling, etc.; and electrochemical cleavage. These methods are further explained in, for example, Sambrook and Ausubel.

In certain embodiments, nucleic acid molecules are obtained from a sample and fragmented for use in the methods disclosed herein. Fragments can be further modified according to any method known in the art and described herein. Nucleic acid fragments can be generated by fragmenting source nucleic acids such as genomic DNA using any method known in the art. In one embodiment, shearing forces during genomic DNA cracking and extraction generate fragments of the desired range. The present invention also encompasses fragmentation methods utilizing restriction endonucleases.

The double-stranded nucleic acid fragments can be any length required for subsequent use, e.g., cloning, transformation, enrichment, sequencing, etc. In certain embodiments, the length of the fragments can be from about 10 to about 50,000 base pairs (bp), and any range therebetween, e.g., from about 100 to about 40,000 bp, from about 300 to 30,000 bp, from about 500 to 20,000 bp, from about 800 to 10,000 bp, from about 1,000 to 8,000 bp, etc. In certain embodiments, the average size of the double-stranded nucleic acid fragments is at least about 100 bp, at least about 200, at least about 300, at least about 500, at least about 1,000, at least about 1,500, at least about 2,000, at least about 5,000, at least about 10,000, at least about 20,000, etc. in length. In some embodiments, the average length of the target nucleic acid sequence inserted into the rolling circle amplification template is between 50 and 20,000 bp, e.g., between 50 and 10,000 bp, between 50 and 5,000 bp, between 50 and 2,000 bp, between 50 and 1,000 bp, between 50 and 500 bp, between 50 and 300 bp, between 50 and 200 bp, between 200 and 800 bp, or between 200 and 500 bp. Thus, in such embodiments, the average length of the forward and reverse target nucleic acid sequences in the resulting concatemers is each between 50 and 20,000 nucleotides, e.g., between 50 and 10,000 nucleotides, between 50 and 5,000 nucleotides, between 50 and 2,000 nucleotides, between 50 and 1,000 nucleotides, between 50 and 500 nucleotides, between 50 and 300 nucleotides, between 50 and 200 nucleotides, between 200 and 800 nucleotides, or between 200 and 500 nucleotides.

In certain embodiments, the fragments are treated to produce blunt ends that are compatible with being connected to adapters having compatible blunt ends. Any suitable method for producing blunt ends can be used, including the use of one or more enzymes having 5' and/or 3' single-stranded exonuclease activity (e.g., E. coli exonuclease III) and/or a fill-in reaction to extend the 3' recessed end (e.g., using T4 DNA polymerase). There is no intention to limit in this regard. In other embodiments, the fragments have sticky ends, such as the ends left by treatment with restriction endonucleases or other nucleases, or 3'A added by Taq polymerase, and the adapters have overhangs complementary to these ends. Obviously, the two ends of the fragment can have different sticky ends, or one end can be blunt and the other end is sticky, and one adapter will be compatible with one end, while the other adapter is compatible with the other end.

Rolling circle amplification

Rolling circle amplification methods that can be used to generate concatemers used as sequencing templates in the present invention from asymmetric circular nucleic acids are well known in the art. See, for example, U.S. Patent No. 5,648,245 to Fire et al., "Concatemer library by RCA," U.S. Patent Application Publication No. 20050069939 to Wang et al., "Amplification of polynucleotides by rolling circle amplification," and U.S. Patent No. 9,290,800 to Turner et al., "Targeted rolling circle amplification," which are incorporated herein by reference in their entirety for all purposes. Rolling circle amplification from oligonucleotides attached to a substrate is described in, for example, U.S. Patent No. 6,274,320 to Rothberg and Bader and U.S. Patent Application Publication No. 20060024711 to Lapidus, which are incorporated herein by reference in their entirety for all purposes.

Nucleic acid sequencing process

Suitable sequencing processes for use in the provided methods include, but are not limited to, sequencing by binding, sequencing by synthesis (sequencing by incorporation), pH-based sequencing, sequencing by polymerase monitoring, sequencing by hybridization, and other methods of massively parallel sequencing or next generation sequencing. Suitable surfaces for sequencing include, but are not limited to, surfaces within a flat substrate, a hydrogel, a nanopore array, a microparticle, a nanoparticle, or a flow cell. Exemplary sequencing platforms including methods, reagents, and solid phase surfaces are described below and in the cited references.

In one aspect, sequencing is performed using sequencing (SBB) technology performed by combining. Exemplary particularly useful sequencing performed by combining reaction is described in the following: U.S. Patent No. 10,077,470, No. 10,443,098, No. 10,400,272 and No. 10,975,427 and U.S. Patent Application Publication No. 20190119742, No. 20180187245 and No. 20200032322, each of which is incorporated herein by reference as a whole. In general, the method for determining the sequence of template nucleic acid molecules by combining sequencing can be based on forming a ternary complex (between polymerase, triggered nucleic acid and homologous nucleotides) under specific conditions. The method may include an inspection phase, followed by a nucleotide incorporation phase.

The inspection phase in sequencing by binding procedures can be performed in a flow cell having at least one template nucleic acid molecule (e.g., a multiplex RCA product) primed with a primer; contacting the primed template nucleic acid molecule with a first reaction mixture comprising a polymerase and at least one nucleotide type; observing the interaction of the polymerase and the nucleotide with the primed template nucleic acid molecule under the condition that the nucleotide is not covalently added to the primer; and using the observed interaction of the polymerase and the nucleotide with the primed template nucleic acid molecule to identify the next base in each template nucleic acid. The interaction between the primed template, the polymerase and the nucleotide can be detected in a variety of schemes. For example, the nucleotide can contain a detectable label. Each nucleotide can have a label that is distinguishable relative to other nucleotides. Alternatively, some or all of the different nucleotide types can have the same label, and the nucleotide types can be distinguished, for example, based on the separate delivery of different nucleotide types or combinations thereof to the flow cell. In some embodiments, the polymerase can be labeled. The polymerase associated with different nucleotide types can have a unique label that distinguishes the nucleotide types associated with them. Alternatively, the polymerases can be similarly labeled, and the different nucleotide types can be distinguished based on separate delivery of the different nucleotide types to the flow cell (eg, a labeled polymerase and one or more unlabeled nucleotides are delivered in combination at one time).

During the inspection phase, the correct nucleotides can be distinguished from incorrect nucleotides by the stability of the ternary complex. A variety of conditions and reagents can be used for the stability of the ternary complex, for example, by preventing the incorporation of nucleotides and/or preventing the dissociation of the ternary complex. For example, the primer can contain a reversible blocking portion that prevents the covalent attachment of the nucleotide, the cofactor required for extension (such as a divalent metal ion) can be absent, the inhibitory divalent cation that inhibits the extension of the polymerase-based primer can be present, the polymerase present in the inspection phase can have a chemical modification and/or mutation that inhibits primer extension, and/or the nucleotide can have a chemical modification that inhibits incorporation, such as removing or changing the 5' modification of the natural triphosphate portion. Conditions such as salt concentration, pH and temperature also contribute to the stability of the ternary complex.

Then the extension phase can be carried out by creating conditions in the flow cell, wherein nucleotides can be added to the primers on each template nucleic acid molecule. In certain embodiments, this relates to removing the reagents used in the inspection phase, and replacing them with reagents that promote extension. For example, the inspection reagent can be replaced with a polymerase and nucleotides that can extend. Alternatively, one or more reagents can be added to the inspection phase reaction to create extension conditions. For example, catalytic divalent cations can be added to the inspection mixture lacking cations, and/or polymerase inhibitors can be removed or disabled, and/or nucleotides with extension ability can be added, and/or unblocking reagents can be added so that the primers have extension ability, and/or a polymerase with extension ability can be added. Optionally, the nucleotides enzymatically incorporated into the primer chain of the triggered template nucleic acid molecule are different from the nucleotides used in the inspection step of identifying the next correct nucleotide.

Optionally, the polymerase used in the incorporation step is different from the polymerase used in the inspection step. Optionally, the incorporated nucleotide is a reversible terminator nucleotide, wherein primer extension is limited to a single nucleotide incorporation prior to removal of the reversible terminator portion. Therefore, for embodiments using reversible terminator nucleotides, the unblocking reagent may be delivered to the flow cell (before or after detection occurs). Washing may be performed between different delivery steps.

The above-mentioned inspection phase and extension phase can be carried out in cycles so that in each cycle a single next correct nucleotide is inspected (i.e., the next correct nucleotide is a nucleotide that correctly binds to a nucleotide in the template nucleic acid, and the nucleotide in the template nucleic acid is located immediately 5' to the base in the template that hybridizes to the 3' end of the hybridization primer), and then, the single next correct nucleotide is added to the primer. Any number of cycles can be performed, including, for example, at least 1, 2, 5, 10, 20, 25, 30, 40, 50, 75, 100, 150 or more cycles. Alternatively or additionally, the number of cycles can be limited to no more than 150, 100, 75, 50, 40, 30, 25, 20, 10, 5, 2 or 1 cycles. The cycle sequencing process generates reads of all or part of the template nucleic acid sequence.

Sequencing by synthesis (SBS) technology can also be used. This technology generally involves enzymatic extension of primers by iteratively adding nucleotides for the template strand hybridized with primers. In short, SBS can be initiated by contacting the target nucleic acid attached to the feature of the flow cell with one or more labeled nucleotides, DNA polymerases, etc. The features of primers extended thereon using the target nucleic acid as a template will be combined with the nucleotides of the label that can be detected. Optionally, the labeled nucleotides can further include reversible termination characteristics, and once nucleotides have been added to the primer, the characteristic terminates further primer extension. For example, nucleotide analogs with reversible terminator parts can be added to the primer so that subsequent extension does not occur until an unblocking agent is delivered to remove the part. Therefore, for an embodiment using reversibility to terminate, an unblocking agent can be delivered to the flow cell (before or after detection occurs). Washing can be performed between different delivery steps. Then the cycle can be repeated. For example, exemplary SBS procedures, reagents, and detection instruments that can be readily adapted for use with concatemer arrays in the methods of the present disclosure are described in Bentley et al., Nature 456:53-59 (2008), WO 04/018497, WO 91/06678, WO 07/123744, U.S. Pat. Nos. 7,057,026, 7,329,492, 7,211,414, 7,315,019, and 7,405,281, and U.S. Patent Application Publication No. 2008/0108082, each of which is incorporated herein by reference. Also useful are SBS methods commercially available from Illumina, Inc. (San Diego, California).

Some SBS embodiments include detecting protons released when nucleotides are incorporated into extension products. For example, sequencing based on detecting released protons can use reagents and electrical detectors commercially available from Thermo Fisher Scientific (Waltham, MA) or described in the following U.S. Patent Application Publications: Nos. 2009/0026082, 2009/0127589, 2010/0137143, and 2010/0282617, each of which is incorporated herein by reference.

Other sequencing procedures can be used, such as pyrophosphate sequencing. Pyrophosphate sequencing detects the release of inorganic pyrophosphate (PPi) when a specific nucleotide is incorporated into a nascent primer hybridized to a template nucleic acid strand. See, for example, Ronaghi et al., Analytical Biochemistry 242 (1), 84-9 (1996); Ronaghi, Genome Res. 11 (1), 3-11 (2001); Ronaghi et al., Science 281 (5375), 363 (1998); and U.S. Pat. Nos. 6,210,891, 6,258,568 and 6,274,320, each of which is incorporated herein by reference. In pyrophosphate sequencing, the released PPi can be detected by being converted into adenosine triphosphate (ATP) by ATP sulfurylase, and the resulting ATP can be detected via photons generated by luciferase. Therefore, the sequencing reaction can be monitored via a luminescence detection system.

Sequencing by ligation is also useful, including, for example, those described in Shendure et al. Science 309: 1728-1732 (2005) and U.S. Pat. Nos. 5,599,675 and 5,750,341, each of which is incorporated by reference. Some embodiments may include sequencing by hybridization procedures, such as described, for example, in Bains et al. Journal of Theoretical Biology 135(3), 303-7 (1988); Drmanac et al. Nature Biotechnology 16, 54-58 (1998); Fodor et al. Science 251(4995), 767-773 (1995); and WO 1989/10977, each of which is incorporated by reference. In sequencing by ligation and sequencing by hybridization procedures, a primer hybridized to a nucleic acid template undergoes repeated cycles of extension by oligonucleotide ligation. Typically, the oligonucleotides are fluorescently labeled and can be detected to determine the sequence of the template.

Some embodiments may utilize methods involving real-time monitoring of DNA polymerase activity. For example, nucleotide incorporation may be detected by fluorescence resonance energy transfer (FRET) interactions or zero-mode waveguides (ZMW) between a polymerase carrying a fluorophore and a γ-phosphate labeled nucleotide. For example, techniques and reagents for sequencing via FRET and/or ZMW detection are described in Levene et al., Science 299, 682-686 (2003); Lundquist et al., Opt. Lett. 33, 1026-1028 (2008); and Korlach et al., Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures of which are incorporated herein by reference.

Paired-end sequencing

As known in the art, double-ended sequencing has many uses. For example, in some cases, by using a combination of sequencing technology or sequencing by synthesis technology, particularly when using blocked labeled nucleotides, the amount of sequence data that can be reliably obtained from the target nucleic acid can be limited to, for example, tens or hundreds of cycles of incorporation (and therefore tens or hundreds of bases in sequencing reads). Although such short reads may be very useful, particularly in applications such as, for example, SNP analysis and genotyping, in many cases, it is advantageous to be able to reliably obtain further sequence data of the same target molecule. The technology of "double-ended" or "paired" sequencing allows two reads of a sequence to be determined from two positions on a single polynucleotide duplex, such as from two opposite ends of a duplex. For example, in applications where the length of a target duplex is more than twice the average sequencing read length, it is known that "double-ended" sequences appear on a single duplex and are therefore connected or paired in the genome, which greatly helps to assemble the entire genome sequence into a consensus sequence. In applications where the length of the target duplex is less than twice the average sequencing read length, reads from opposite ends overlap in the middle of the target and can therefore be compared to increase the accuracy of base calls in that region, which is useful because accuracy decreases as sequencing technologies approach their read length limits. Additional uses for paired-end sequences are well known in the art.

An improved method for double-end sequencing is provided herein. The following references provide alternative methods and uses for double-end sequencing. See, for example, U.S. Patent No. 8,192,930 to Vermass, "Method for sequencing a polynucleotide template", U.S. Patent No. 8,105,784 to Rigatti, "Paired end reads on libraries made by bridge amplification", Chen's International Patent Application Publication WO2004070005 "Double ended sequencing by blocking and unblocking", and U.S. Patent Application Publication No. 20210189483 "Controlled strand displacement for paired-end sequencing", which are incorporated herein by reference in their entirety for all purposes.

Compositions, systems and kits

Compositions, systems and kits related to, produced by or used in these methods are also features of the present invention. For example, a general class of embodiments provides a composition comprising an array of nucleic acid concatemers. The concatemers are bound to a surface, for example, wherein different concatemers are at different sites in an orderly arrangement, or different concatemers are at different sites in an unordered array. The position of a given concatemer in the array can be predetermined or random. Each concatemer includes multiple copies of: a first adapter region including a first sequencing primer binding site, a forward strand of a target nucleic acid sequence, a second adapter region different from the first adapter region, and a reverse strand of a target nucleic acid sequence complementary to the forward strand. The second adapter region may include a second sequencing primer binding site having a sequence different from the first sequencing primer binding site.

Concatemers can be covalently or non-covalently bound to the surface. For example, each concatemer can include a first member of a binding pair (e.g., biotin), which is combined with a second member of a binding pair (e.g., avidin or streptavidin) that is in turn bound to the surface. The array can include a large number of concatemers, thousands to millions to billions (e.g., at least one million, at least ten million, at least one hundred million, at least one billion, at least two billion or at least three billion concatemers). The concatemers in the array can be fixed on a flat surface of, for example, a substrate, on a non-flat surface of the substrate, or fixed in a three-dimensional manner in the substrate, such as in a gel matrix. Suitable substrates are well known in the art, and include but are not limited to the surface of a slide, a chip, a flow cell, etc.

In some embodiments, the first sequencing primer is hybridized to the first sequencing primer binding site. In some embodiments, the second sequencing primer is hybridized to the second sequencing primer binding site. The composition may include a nascent chain generated by extending the first sequencing primer and/or the second sequencing primer. The nascent chain generated by the extension of the first sequencing primer is optionally blocked. The composition optionally further includes: a polymerase (e.g., a strand displacement polymerase or a polymerase lacking strand displacement activity), one or more nucleotides (e.g., naturally occurring nucleotides, non-natural nucleotides, labeled nucleotides, reversible terminator nucleotides and/or chain termination nucleotides), a masked primer, a blocking oligonucleotide, a displacement primer, a masked strand, a displacement strand, one or more stapled oligonucleotides and/or other reagents used in the sequencing process. The composition is optionally present in a nucleic acid sequencing system.

Essentially all of the features described above are also relevantly applicable to these embodiments, for example, regarding the copy number of the repeat unit in the concatemer, the inclusion of nucleases in the composition for removing nascent strands or masking strands or nicking the linker region, the inclusion of single-stranded binding proteins in the composition, suitable array substrates, etc.

Another general class of embodiments provides a kit comprising any combination of one or more of the following: a solid support configured to bind multiple nucleic acid concatemers, a first stem-loop adapter, a second stem-loop adapter different from the first stem-loop adapter, reagents for performing rolling circle amplification (e.g., rolling circle amplification primers, strand displacement polymerases, and one or more nucleotides), a first sequencing primer, optionally a second sequencing primer, and reagents for performing nucleic acid sequencing (e.g., polymerases and one or more nucleotides, optionally containing one, two, three, or four labeled nucleotides). The polymerases used for rolling circle amplification and sequencing are typically different polymerases, but may be the same in some embodiments. The kit may also include additional reagents for generating circular nucleic acid molecules, performing rolling circle amplification to generate concatemers, and performing nucleic acid sequencing, including but not limited to a buffered reaction solution, a masking primer, a blocking oligonucleotide, a displacement primer, one or more stapled oligonucleotides, a site-specific endonuclease, and/or an exonuclease. The kit typically also includes instructions for using the components to perform the desired process also described or referenced herein, for example, for generating circular nucleic acid molecules, performing rolling circle amplification to generate concatemers, and performing nucleic acid sequencing. The components of the kit are packaged in one or more containers.

Essentially all features described above also apply to these embodiments, e.g. with respect to suitable array substrates, inclusion of single-stranded binding proteins, etc.

Various nucleic acid sequencing systems suitable for performing sequencing processes useful in the methods of the present invention are known in the art (see, e.g., the references above detailing sequencing techniques) and/or are commercially available. Such sequencing systems may include a fluid handling system (e.g., for delivering reagents to a substrate including a concatemer array during the sequencing process) and a detection system.

For example, a sequencing process (e.g., a sequencing process using the substrate described above and the compositions and methods of the present invention) can be developed and utilized in the context of a fluorescent optical system that is capable of irradiating various locations on the substrate and obtaining, detecting, and separately recording fluorescent signals from these locations (e.g., each individually distinguishable location occupied by a different multiplex template). Such systems typically use one or more illumination sources to provide excitation light of appropriate wavelengths for the marker being used. The optical system directs the excitation light to the reaction area, collects the emitted fluorescent signal and directs it to an appropriate one or more detectors. Additional components of the optical system can provide separation of spectrally different signals (e.g., from different fluorescent labels), and direct these separated signals to different parts of a single detector or different detectors. Other components can provide spatial filtering of optical signals, focusing and directing the excitation and/or emission light to the substrate, and focusing and directing from the substrate.

Method described herein can further comprise computer-implemented process, and/or be attached to the software of instructing such process on computer-readable medium.Therefore, the signal data generated by reaction described above and optical system are input or otherwise received in computer or other data processor, and experience one or more in each processing step or assembly.Once these processes have been carried out, the output of the computer-implemented process obtained can be produced in a tangible or observable format, for example, printed in a user-readable report or displayed on a computer display.The output obtained can be stored in one or more databases, for later assessment, processing, reporting, etc., or it can be retained by a computer or transferred to different computers, for configuring subsequent reaction or data processing.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes therefrom will be suggested to those skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.

Although the aforementioned invention has been described in some detail for the purpose of clarity and understanding, it will be clear to those skilled in the art, by reading this disclosure, that various changes in form and detail may be made without departing from the true scope of the invention. For example, all techniques and equipment described above may be used in various combinations. All publications, patents, patent applications, and/or other documents cited in this application are incorporated by reference in their entirety for all purposes, to the extent that each individual publication, patent, patent application, and/or other document is individually indicated to be incorporated by reference for all purposes, including for the purpose of describing and disclosing devices, compositions, preparations, and methods described in publications and that may be used in conjunction with the presently described invention.

Claims (43)

1. A method for double ended sequencing, comprising:

a) Providing a nucleic acid concatemer comprising multiple sequential copies of: a first adapter region, a forward strand of a target nucleic acid sequence, a second adapter region different from the first adapter region, and a reverse strand of the target nucleic acid sequence complementary to the forward strand; and

B) Performing a sequencing process to generate double-ended reads of the target nucleic acid sequence by:

i) Hybridizing a first sequencing primer to the first adapter region and obtaining a first read of a first portion of the target nucleic acid sequence by sequencing from the first sequencing primer; and

Ii) hybridizing a second sequencing primer to the second adapter region and obtaining a second read of a second portion of the target nucleic acid sequence by sequencing from the second sequencing primer;

Wherein the first read and the second read constitute a double-ended read of the target nucleic acid sequence.

2. The method of claim 1, wherein the nucleic acid concatemers are generated by:

Providing a circular nucleic acid molecule comprising: a central region comprising the forward strand and the complementary reverse strand; the center region has two ends, the forward chain is connected to the reverse chain at one end with a first connection region and the forward chain is connected to the reverse chain at the other end with a second connection region; and

Rolling circle amplification is performed using the circular nucleic acid molecule as a template to produce the nucleic acid concatemer.

3. The method of claim 2, wherein the rolling circle amplification step is performed in solution.

4. The method of claim 2, wherein the rolling circle amplification step is performed on the surface of a solid support.

5. The method of claim 1, wherein step ii) is performed after step i).

6. The method of claim 5, wherein after the first read is obtained, nascent strand formed by sequencing from the first primer is removed prior to hybridizing the second sequencing primer to the second adapter region.

7. The method of claim 6, wherein the nascent strand formed by sequencing from the first primer is removed by cleavage and washing, exonuclease digestion, or denaturation.

8. The method of claim 5, wherein the nascent strand formed by sequencing from the first primer is not removed.

9. The method of claim 8, wherein the 3' end of the nascent strand formed by sequencing from the first primer is blocked prior to hybridizing the second sequencing primer to the second adapter region.

10. The method of claim 1, wherein step i) and step ii) are performed simultaneously.

11. The method of claim 10, wherein sequencing from the first sequencing primer and the second sequencing primer to obtain the first read and second read comprises detecting a signal, wherein a signal conducive to obtaining the first read is distinguishable from a signal conducive to obtaining the second read based on its intensity.

12. The method of claim 11, wherein the first sequencing primer and the second sequencing primer are provided at different concentrations.

13. The method of claim 11, wherein the second sequencing primer is provided in the form of a mixture of an extendable oligonucleotide and a non-extendable oligonucleotide.

14. The method of claim 11, wherein the first sequencing primer and the second sequencing primer anneal to their respective adapter regions with significantly different efficiencies.

15. The method of claim 10, wherein sequencing from the first sequencing primer and the second sequencing primer to obtain the first read and the second read comprises mapping to a known reference sequence.

16. The method of claim 1, wherein sequencing from the first sequencing primer and the second sequencing primer comprises extending the first sequencing primer and the second sequencing primer with a strand displacement polymerase.

17. The method of claim 1, wherein sequencing from the first sequencing primer and the second sequencing primer is performed in the presence of a single strand binding protein.

18. The method of claim 1, wherein performing a sequencing process to generate a double-ended read of the target nucleic acid sequence comprises: synthesizing a first masking strand complementary to the forward strand prior to hybridizing the first sequencing primer to the first adapter region, and synthesizing a second masking strand complementary to the reverse strand prior to hybridizing the second sequencing primer to the second adapter region.

19. The method of claim 1, wherein sequencing from the first sequencing primer and the second sequencing primer comprises sequencing by a binding technique.

20. The method of claim 1, wherein the nucleic acid concatemer comprises at least 100 sequential copies of the first adapter region, the forward strand, the second adapter region, and the reverse strand.

21. A method for nucleic acid sequencing, comprising:

Providing a nucleic acid concatemer comprising multiple sequential copies of: a first adapter region, a forward strand of a target nucleic acid sequence, a second adapter region different from the first adapter region, and a reverse strand of the target nucleic acid sequence complementary to the forward strand;

Hybridizing a masking primer to the second adapter region and extending the masking primer to produce a first masking strand complementary to the forward strand, wherein the first masking strand is not equally complementary to the entire first adapter region; and

A first sequencing primer is hybridized to the first adapter region and sequenced from the first sequencing primer to obtain a first read of the first portion of the target nucleic acid sequence.

22. The method of claim 21, wherein extending the masking primer comprises extending the masking primer with a strand displacement polymerase.

23. The method of claim 22, comprising hybridizing an oligonucleotide that blocks strand displacement to the first adapter region prior to extending the masking primer.

24. The method of claim 21, wherein the first adapter region comprises at least one non-natural nucleotide, wherein the masking primer extends under conditions that exclude the complement of the at least one non-natural nucleotide.

25. The method of claim 21, comprising introducing a cut into the first adapter region prior to extending the masking primer.

26. The method of claim 25, comprising hybridizing one end of a staple-type oligonucleotide to the first adapter region and hybridizing the other end of the staple-type oligonucleotide to the second adapter region.

27. The method of claim 25, wherein the 5' end of the first sequencing primer hybridizes to the second adapter region.

28. The method of claim 25, wherein the 5' end of the masking primer hybridizes to the first adapter region.

29. The method as claimed in claim 21, comprising:

After sequencing from the first sequencing primer, further extending the nascent strand produced by sequencing from the first sequencing primer to produce a second masked strand complementary to the reverse strand, wherein the second masked strand is not also complementary to the entire second adapter region;

Removing the first masking chain; and

Hybridizing a second sequencing primer to the second adapter region and obtaining a second read of a second portion of the target nucleic acid sequence by sequencing from the second sequencing primer.

30. The method of claim 29, wherein the masking primer comprises a 5' phosphate group, and wherein removing the first masking strand comprises digesting the first masking strand with lambda exonuclease.

31. The method of claim 21, wherein the first sequencing primer comprises a5 'region and a 3' region that each hybridizes to the first adapter region and flank a central region that does not hybridize to the first adapter region, wherein a portion of the first adapter region remains single stranded when the first sequencing primer hybridizes to the first adapter region; the method comprises the following steps:

Further extending the nascent strand generated by sequencing from the first sequencing primer to generate a first extended strand complementary to the reverse strand, wherein the first extended strand is not likewise complementary to the entire second adapter region;

hybridizing a displacement primer to a single stranded portion of the first adapter region;

Extending the displacement primer with a strand displacement polymerase to displace the first extension strand from the reverse strand, wherein the 5' region of the first extension strand remains hybridized to the first adapter region; and

A second sequencing primer is then hybridized to the first extended strand, and a second read of the second portion of the target nucleic acid sequence is obtained by sequencing from the second sequencing primer.

32. The method of claim 21, wherein the nucleic acid concatemers are generated by:

Providing a circular nucleic acid molecule comprising: a central region comprising the forward strand and the complementary reverse strand; the center region has two ends, the forward chain is connected to the reverse chain at one end with a first connection region and the forward chain is connected to the reverse chain at the other end with a second connection region; and

Rolling circle amplification is performed using the circular nucleic acid molecule as a template to produce the nucleic acid concatemer.

33. The method of claim 21, wherein the rolling circle amplification step is performed in solution.

34. The method of claim 21, wherein the rolling circle amplification step is performed on the surface of a solid support.

35. The method of claim 21, wherein sequencing from the first sequencing primer comprises sequencing by a binding technique.

36. A composition, comprising:

An array of nucleic acid concatemers, each nucleic acid concatemer bound to a surface and comprising multiple copies: a first adapter region comprising a first sequencing primer binding site, a forward strand of a target nucleic acid sequence, a second adapter region different from the first adapter region, and a reverse strand of the target nucleic acid sequence complementary to the forward strand.

37. The composition of claim 36, wherein the second adapter region comprises a second sequencing primer binding site.

38. The composition of claim 36, comprising a first sequencing primer that hybridizes to the first sequencing primer binding site.

39. The composition of claim 36, comprising a second sequencing primer that hybridizes to the second sequencing primer binding site.

40. The composition of claim 36, comprising a polymerase.

41. The composition of claim 36, comprising one or more of a masking primer, a blocking oligonucleotide, a displacement primer, a masking strand, and a displacement strand.

42. The composition of claim 36, wherein the composition is present in a DNA sequencing system.

43. A kit, comprising:

A solid support configured to bind a plurality of nucleic acid concatemers;

a first stem-loop adapter;

A second stem-loop adaptor different from the first stem-loop adaptor;

Reagents for performing rolling circle amplification;

A first sequencing primer; and

Reagents for performing nucleic acid sequencing.