Recovery phase in massively parallel sequencing

Abstract

Determining the sequence of a nucleic acid typically requires multiple cycles of reactions that generate a signal based on the identity of one or more nucleotides in the sequence. Sequencing is typically performed on multiple copies of a template to enhance the signal and improve accuracy. However, as the number of cycles increases, some copies become out of phase, increasing the signal-to-noise ratio and compromising accuracy. The present invention provides a strategy that uses blocking groups and dinucleotide recognition to make each copy in phase. This improves accuracy and enables the user to increase the length of the sequence read.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 991,440, filed on March 18, 2020. The entire contents of these applications are incorporated herein by reference for all purposes. Technical Field

[0003] The techniques of the present disclosure generally relate to determining the nucleic acid sequence of a target polynucleotide, such as genomic DNA. Background Art

[0004] The demand for low-cost, high-throughput methods for nucleic acid sequencing and resequencing led to the development of "massively parallel sequencing" (MPS) technology.

[0005] A commonly used method for DNA sequencing is called "sequencing by synthesis" (SBS), as disclosed in Ronaghi et al., Science, 281:363-365, 1998; Li et al., Proc. Natl. Acad. Sci. USA, 100:414-419, 2003; Metzker, Nat Rev Genet. 11:31-46, 2010; Ju et al., Proc. Natl. Acad. Sci. USA 103:19635–19640, 2006; Bentley et al., Nature 456:53–59, 2008; and in U.S. Pat. Nos. 6,210,891, 6,828,100, 6,833,246 and 6,911,345 and 10,190,162. SBS typically requires the extension of a primer hybridized to a single-stranded template polynucleotide by the controlled incorporation of the correct complementary nucleotide opposite to the template being sequenced. The resulting product is sometimes referred to as an "extended primer" or "growing chain." In one method, a reversible terminator nucleotide (RT) is used to determine the sequence of the DNA template. In the most commonly used SBS method, each RT includes a modified nucleotide that includes: (1) a blocking group that ensures that only a single base is added to the 3" end of the growing DNA copy chain by a DNA polymerase, and (2) a fluorescent label. An alternative method, called Sequencing, wherein unlabeled reversible terminator nucleotides are used, both of which are incorporated herein by reference. Summary of the invention

[0006] In some massively parallel sequencing (MPS) methods, determining the sequence of a nucleic acid typically requires (i) preparing a library of different template sequences, (ii) fixing many copies of each template sequence ("clonal population") at different sites on a substrate, and (iii) performing multiple cycles of sequencing reactions. The different sites on the substrate can be located at random, spaced-apart positions, or the sites can be arranged in an ordered array. The sequencing reaction produces a signal at each position on the array. The signal is produced by a method that includes incorporating nucleotides into each copy of the template. Sequencing the clonal population at each site (rather than a single template molecule) results in a stronger signal, improves accuracy, and in some methods, reduces amplification errors that occur in the process of generating the clonal population.

[0007] However, these advantages require the incorporation of complementary nucleotides at each position that occurs in phase in each cycle. That is, in each cycle in which nucleotides are incorporated, incorporation occurs in most or nearly all copies of the template at that position on the array. However, in each cycle, for a certain number of templates in the clonal population at that site, non-incorporation or misincorporation may occur. Unincorporated or misincorporated templates may be out of phase with other templates at that site. As the number of cycles increases, out-of-phase templates accumulate at each site, increasing the signal-to-noise ratio, reducing accuracy, and limiting the read length of these sites. The present disclosure describes a re-phasing strategy for reducing the number of out-of-phase templates, resulting in improvements in accuracy and read length.

[0008] Examples of clonal populations of template sequences include, for purposes of illustration and not limitation, DNA nanoballs (single-stranded concatemers having many copies of the template sequence and adapter sequences including primer binding sites), in situ amplification products including DNBs), double-stranded DNA concatemers, clonal clusters of amplicons generated by bridge amplification or other amplification methods.

[0009] In general, the present disclosure provides a method for re-phasing extension primers in a clonal population of nucleic acid duplexes, the clonal population of nucleic acid duplexes comprising extension primers hybridized to a template sequence, wherein a plurality of extension primers in the clonal population have different 3' ends and are therefore out of phase. This can be achieved by the following steps: (1) extending the extension primers by incorporating one or more nucleotides complementary to the template sequence and nucleotides comprising nucleotide triphosphates A, T, C and G or their analogs using a polymerase, wherein one of the nucleotides is a reversible terminator blocked by a first blocking group and the other three nucleotides are not blocked until substantially all of the extension primers are blocked; and then (2) unblocking the extension primers.

[0010] The general method includes a dinucleotide frequency re-phasing (DFR) technique. Each extension primer is extended until a selected dinucleotide is reached, wherein the selected dinucleotide has the formula XY. The first nucleotide of the dinucleotide (X) can be a reversible terminator blocked by a first blocking group, and Y is the second nucleotide of the dinucleotide. The second nucleotide (Y) can be a reversible terminator blocked by a second blocking group, or a blocked denatured oligonucleotide containing Y, for example, at the 5' end.

[0011] In a general DFR method, an extension primer in a clonal population of nucleic acid duplexes is re-phased to a selected dinucleotide (XY), wherein each of the duplexes comprises an extension primer annealed to a template sequence. The method is useful when the extension primers in the clonal population have different 3' ends and are therefore out of phase. The re-phasing method is accomplished by performing the following multiple cycles: (i) extending the extension primer using a first mixture comprising a polymerase and four nucleotide triphosphates selected from A, T, C, and G and / or their analogs, wherein one of the nucleotide triphosphates or analogs in the first mixture corresponds to the first nucleotide (X) of the selected dinucleotide and is blocked by a first blocking group, and wherein the other three nucleotide triphosphates or analogs in the first mixture are not blocked, and extension continues until substantially all extension primers are blocked by the first blocking group.

[0012] Subsequent steps include (ii) unblocking the first blocking group; and (iii) treating the extended primer from (ii) with a second mixture comprising a polymerase and a single nucleotide triphosphate selected from A, T, C, and G and analogs thereof corresponding to a second nucleotide (Y) of the selected dinucleotide and blocked by a second blocking group. The second mixture optionally includes three nucleotide triphosphates or analogs that do not correspond to the second nucleotide (Y) blocked by the first blocking group (or, in some embodiments, are blocked by a plurality of first blocking groups that do not include a second blocking group), for example, T*C**G*** is the second blocking group, *, **, and *** are the first blocking groups, and the second blocking group is not unblocked under the same conditions as the unblocked blocking group-1. The plurality of cycles are repeated until substantially all of the extension primers are blocked by the second blocking group. The second blocking group is then unblocked, thereby re-phasing the extension primers in the clonal population.

[0013] In a kind of such method, the only nucleotide triphosphates in the second mixture are nucleotide triphosphates or analogs sealed by the second blocking group. Or, the second mixture comprises nucleotide triphosphates or analogs sealed by the second group, and also comprises trinucleotide triphosphates or analogs that do not correspond to the second nucleotide (Y) sealed by the first blocking group. Any one of the first and second blocking groups can be an O-azidomethyl, and the other one of the first and second blocking groups can be an O-NH2 group.

[0014] Another variant of the general method of DFR uses an oligonucleotide to identify the second nucleotide. This is accomplished by performing multiple cycles of the following steps: (i) extending the extension primer using a first mixture comprising a polymerase and four nucleotide triphosphates selected from A, T, C, and G and / or its analogs, wherein one of the nucleotide triphosphates or analogs in the first mixture corresponds to the first nucleotide (X) of the selected dinucleotide and is blocked by a first blocking group, and wherein the other three nucleotide triphosphates or analogs in the first mixture are unblocked, and the extension continues until substantially all extension primers are blocked by the first blocking group; then (ii) the first blocking group is unblocked; and (iii) the extension primer is treated with a second mixture comprising a ligase and a 5' phosphorylated oligonucleotide. The oligonucleotide is blocked at the 3' end, wherein the base in the oligonucleotide corresponds to the second nucleotide (Y) of the selected dinucleotide. Repeating the multiple cycles again, until substantially all extension primers are blocked by the oligonucleotide; therefore, the oligonucleotide is unblocked, thereby re-phasing the extension primers in the clonal population.

[0015] The 5' phosphorylated oligonucleotide optionally has the formula 5'-phos-B(N) Z -X, wherein "5'-phos" is a phosphorylated nucleotide, B is a nucleotide defining a ligation site, X is a blocking structure (i.e., a structure that prevents polymerase-mediated extension of the oligonucleotide), which may be an irreversible blocking structure, Z is 6-20, preferably 6-15, more preferably 6-12, and (N) Z Is a degenerate oligonucleotide sequence. In some embodiments, Z is 9. In some embodiments, B is uracil (U). Unblocking may include removing the entire oligonucleotide from the extension primer. Optionally, the irreversible blocking structure in the oligonucleotide is incorporated by reverse dT (IDT) at the 3'-end, thereby generating a 3'-3' bond, which inhibits both the degradation of 3' exonucleases and the extension of DNA polymerases. When B is uracil, the 5' phosphorylated oligonucleotide can be unblocked by treating with an enzyme mixture of uracil-DNA glycosylase (UDG) and apurinic / apyrimidinic endonuclease 1 (Apel) to cut and remove the uracil base. Typically, in any dinucleotide phasing method, 5-15 cycles are performed.

[0016] To avoid creating gaps in the sequence read during rephasing, the user may wish to remove 5-50 bases from the 3' end of each primer prior to rephasing, thereby realigning the 3' end of the extension primer to an upstream position. This can be accomplished in several different ways: for example, during the synthesis sequencing process performed prior to rephasing, including during at least some cycles of sequencing, uracil triphosphates or their analogs can be incorporated into the extension primers in place of thymine triphosphates; the extension primers are then cleaved at the incorporated uracil bases. Cleavage can be performed using an enzyme mixture of uracil-DNA glycosylase (UDG) and apurinic / apyrimidinic endonuclease 1 (Apel).

[0017] Another way to adjust the 3' end of the upstream extension primer is as follows: during synthesis sequencing prior to re-phasing, including in at least some cycles of sequencing, nucleotide triphosphates containing ribonucleotides (RNA) or 5' α-phosphothioate modified nucleotides; then cleaving the extension primer at the incorporated RNA base or the incorporated 5'-α-phosphothioate modified nucleotide. Alternatively, re-adjustment includes treating the extension primer with a 3' exonuclease under controlled conditions, or treating the extension primer with a subsequence-dependent nicking enzyme, thereby removing the 5-50 bases from the 3' end of the extension primer.

[0018] After the above-mentioned re-phasing method, the sequencing cycle can be resumed, whereby the extension primers in the clonal population are extended by bases, each base recognizing a complementary nucleotide in the template sequence. By performing multiple sequencing cycles, long sequencing reads can be obtained from a clonal population of nucleic acid duplexes (each duplex includes an extension primer that anneals to the template sequence), wherein the extension primer in each duplex is extended by one nucleotide, thereby recognizing a complementary nucleotide in the template sequence. After many such sequencing cycles, the extension primers are re-phased as described above, and then the sequencing cycle is resumed to recognize other nucleotides in the template sequence.

[0019] Rephasing can be performed once, twice, or more as needed to reduce the inconsistency between primers in the clonal population: typically 2-4 times within the first 800 sequencing cycles. The benefits of this include extending the number of clonal populations with inconsistency percentages less than 5%, 2%, or 1% by at least 2, 5, or more times, or by at least 100, 200, or 400 cycles.

[0020] The invention also provides a sequencing method comprising providing an array comprising a plurality of clonal populations, each clonal population comprising a nucleic acid duplex in which a sequencing primer anneals to a template sequence. A plurality of sequencing cycles are performed (e.g., by sequencing by synthesis) to extend the sequencing primers until at least some of the primers have a different 3' end than other primers in the same clonal population and are therefore out of phase with other primers in the clonal population. The sequencing primers are then re-phased using any of the above methods and optional features.

[0021] Rephasing is performed as often as needed or desired during the sequencing process: for example, 2-4 times during the sequencing process to obtain a read length of at least 800 bases, optionally at least 1200, 1600 or 2000 bases.

[0022] A method for re-phasing multiple copies of a nucleic acid to be sequenced, wherein each copy comprises a single-stranded nucleic acid template hybridized to an extendable oligonucleotide sequencing probe, the method comprising:

[0023] (1) extending the probe in a complementary manner to the template using a polymerase and a mixture of nucleotide triphosphates, one of which is a 3′ reversibly blocked nucleotide triphosphate, until substantially all copies are blocked; and then

[0024] (2) Unblocking the reversibly blocked nucleotide incorporated into the extension probe.

[0025] In one aspect, the invention provides a method for re-phasing extended sequence primers hybridized to multiple copies of a nucleic acid template for template sequencing, wherein the primers hybridized to at least some of the copies have different 3' ends and are therefore out of phase with the primers hybridized to other copies of the template, the method comprising: (1) further extending the primers hybridized to each copy by one or more bases complementary to the template using a polymerase and a mixture of nucleotide triphosphates containing A, T, C and G, wherein one of the nucleotide triphosphates is a 3' reversibly blocked nucleotide triphosphate and the other three nucleotide triphosphates are unblocked, until substantially all of the primers are blocked; and then (2) unblocking the reversibly blocked nucleotides incorporated into the primers.

[0026] In one aspect, a method for rephasing is provided, which is a method of dinucleotide frequency rephasing (DFR), wherein each primer is extended until a selected dinucleotide is reached. In one aspect, a method of dinucleotide frequency rephasing (DFR) according to claim 2 is provided, comprising: (a) performing a plurality of rephasing cycles, wherein each cycle comprises the following steps: further extending the primer hybridized to each copy by one or more bases complementary to the template using a first mixture comprising a polymerase and nucleotide triphosphates A, T, C, and G, wherein one nucleotide triphosphate in the first mixture represents the first nucleotide of the selected dinucleotide and is blocked by a first blocking group, and wherein the other three nucleotide triphosphates in the first mixture are unblocked, and the extension continues until substantially all primers are blocked; and then allowing the first blocking group to block the primer hybridized to each copy by one or more bases complementary to the template. (b) after completing all multiple cycles of step (a), unblocking the second blocking group; thereby re-phasing multiple copies, wherein the 3' end of the primer hybridized to each copy is a dinucleotide. In some cases, the method of claim 3, wherein the only triphosphate nucleotides in the second mixture are triphosphate nucleotides blocked by the second blocking group. In some cases, the method of claim 3, wherein the second mixture comprises a nucleotide triphosphate blocked by the second group, and the other three nucleotide triphosphates blocked by the first blocking group. In some cases, the method of any one of claims 3-5, wherein the first blocking group is an O-azidomethyl or ONH2 group, and the second blocking group comprises a disulfide bond.

[0027] In some cases, the nucleotide triphosphate blocked by the first blocking group is C, the nucleotide triphosphate blocked by the second blocking group is A, and the dinucleotide is CA. In some cases, the method of dinucleotide frequency re-phasing (DFR) according to claim 2 comprises: (a) performing multiple re-phasing cycles, wherein each cycle comprises the following steps: using a first mixture comprising a polymerase and triphosphates of nucleotides A, T, C and G, further extending the primer hybridized to each copy by one or more bases complementary to the template, wherein one of the triphosphates in the first mixture represents the first nucleotide of the selected dinucleotide and is blocked by a first blocking group, and wherein the other three nucleotide triphosphates in the first mixture are unblocked, and the extension continues until substantially all primers are blocked; then unblocking the first blocking group; and treating the primer with a second mixture comprising a ligase and a 5'-phosphorylated oligonucleotide, wherein the 5'-phosphorylated oligonucleotide is blocked at the 3' end by a second blocking group, wherein the base in the oligonucleotide represents the second nucleotide of the selected dinucleotide, and wherein the treatment causes the oligonucleotide to be ligated only to the primer adjacent to the portion of the template complementary to the oligonucleotide; (b) after completing all multiple cycles of step (a), unblocking the oligonucleotide. In some cases, the method of claim 8, wherein the 5' phosphorylated oligonucleotide has the formula BN115X, wherein B is a nucleotide base representing the second nucleotide of the selected dinucleotide, each N is a nucleotide homolog or a nucleotide mixture containing nucleotides that can hybridize to any base in the template; X is an irreversible blocking structure; and wherein the unblocking in step (b) comprises removing the oligonucleotide from the primer.

[0028] In some cases, the method of claim 9, wherein the irreversible blocking structure is an inverted dT (IDT) incorporated at the 3'-end of the oligonucleotide, thereby generating a 3'-3' bond that inhibits both degradation by a 3' exonuclease and extension by a DNA polymerase. In some cases, the method of claim 9, wherein B is uracil, and wherein the 5-phosphorylated oligonucleotide is unblocked by treatment with an enzyme mixture of uracil DNA glycosylase (UDG) and apurinic / apyrimidinic endonuclease 1 (Apel) to cleave and remove the uracil base.

[0029] In general, the re-phasing method of the present disclosure comprises the following steps: (1) extending a primer in a complementary manner to a template using a polymerase and a mixture of nucleotide triphosphates, one of which is a 3' reversibly blocked nucleotide triphosphate, until substantially all copies are blocked; and then (2) unblocking the reversibly blocked nucleotide incorporated into the extended primer. In its basic form, re-phasing can be performed using a single blocked nucleotide ("Method One", explained in more detail below).

[0030] The variant included in the re-phasing strategy is dinucleotide frequency re-phasing (DFR) ("method two"). Here, re-phasing does not occur on a single base, but on a selected nucleotide duplex. The method includes performing multiple re-phasing cycles, wherein each cycle includes the following steps: using a polymerase and a first mixture of nucleotide triphosphates to extend the primer in a complementary manner to the template, wherein one of the nucleotide triphosphates in the first mixture is blocked by a first blocking group until substantially all copies are blocked. The first blocking group is then unblocked, and a polymerase and a second mixture of nucleotide triphosphates are used to extend the primer with a single nucleotide (the second nucleotide of the duplex) in a complementary manner to the template, wherein a triphosphate nucleotide in the second mixture is blocked by a second blocking group, and the remaining nucleotides are blocked by the first blocking group. When the re-phasing cycle is completed, the second blocking group is subsequently unblocked to complete the process.

[0031] In the context of DFR, the terms "dinucleotide", "2mer" and "duplex" are used interchangeably. Any combination of two nucleotides can be used, including nucleotide repeats, for a total of 16 possible choices. A better choice is CA, where the nucleotide blocked by the first blocking group is C and the nucleotide blocked by the second blocking group is A. Some other duplexes may be less preferred due to their lower frequency in the human genome. This means that more re-phasing steps may be needed to achieve the same degree of consistency.

[0032] In the second mixture in the DFR process, one of the nucleotides is blocked by the second blocking group, and the other nucleotides may also be blocked by the first blocking group. In this case, the last step includes unblocking the first and second blocking groups. As an example, the first blocking group may be an adjacent azidomethyl group, and the second blocking group may include an ONH2 group.

[0033] Another re-phasing method is oligonucleotide re-phasing ("Method 4"). The method includes performing multiple re-phasing cycles, wherein each cycle includes: extending the primer in a complementary manner to the template using a polymerase and a mixture of nucleotide triphosphates, wherein one of the nucleotide triphosphates is a nucleotide triphosphate blocked by a first blocking group, until substantially all copies are blocked; then unblocking the first blocking group; and extending the primer in a complementary manner to the template by connecting a 5' phosphorylated oligonucleotide to a sequencing primer, wherein the 5' phosphorylated oligonucleotide is blocked at the 3' end with a second blocking group. After the cycle is completed, the re-phasing is completed by unblocking the 5' phosphorylated oligonucleotide.

[0034] In one approach, the phosphorylated oligonucleotide has the formula U(N) Z-X, wherein U is uracil, X is a closed structure, Z is 6-20, and (N) Z is a degenerate oligonucleotide and X is an irreversible blocking structure. The irreversible blocking structure can be incorporated with an inverted dT (IDT) at the 3' end of the oligonucleotide, thereby generating a 3'-3' bond that inhibits both degradation by 3' exonucleases and extension by DNA polymerases. The 5' phosphorylated oligonucleotide can be unblocked by treating with an enzyme cocktail of uracil-DNA glycosylase (UDG) and apurinic / apyrimidinic endonuclease 1 (Apel) or similar abasic site endonucleases to cleave and remove uracil bases.

[0035] Using any of these methods, more cycles or rounds of rephasing increase the number of clonal populations in which growing chains are greater than 90% in phase or greater than 99% in phase. Typically, a rephasing module comprises 5 to 15 or 5 to 10 rephasing rounds or cycles, such as 5, 6 or 7 rounds.

[0036] Any of these re-phasing methods can optionally be performed after realigning the site back to the strand being sequenced. This trims the most recent base added to the primer and provides a degree of sequence overlap. If this is done, the realignment or back-off is typically 5-50 bases, usually at least 10, 20 or 30 bases.

[0037] One way in which re-adjustment can be performed is to add uracil residues at some sequencing step prior to re-phasing, with a length corresponding to the cutback window. The primer is extended in a complementary manner to the template using a polymerase and a mixture of nucleotide triphosphates, one of which is uracil. The primer is then cleaved at the incorporated uracil base: for example, using an enzyme mixture of uracil-DNA glycosylase (UDG) and apurinic / apyrimidinic endonuclease 1 (Apel) or a similar abasic site endonuclease.

[0038] Another method to re-adjust the sequencing site involves extending the primer in a complementary manner to the template using a polymerase and a mixture of nucleotide triphosphates, one of which is an RNA base or a thiolated nucleotide; and then cleaving the primer at the incorporated RNA base. Another method to re-adjust the site involves treating the primer with a controlled 3' exonuclease or with a nicking enzyme in a sequence-dependent manner.

[0039] When re-phasing is complete, the copies of the target fragment in each amplicon are typically at least 90% in phase, preferably 97%, 99% or substantially 100% in phase. The number of re-phased amplicons that are 100% in phase can be at least 70%, 80%, 90% or 95% of the processed amplicons, as shown in the figure.

[0040] After re-phasing, the base-by-base sequencing cycle can be resumed. Thus, the technology provided by the present disclosure can be used to obtain long sequencing reads from multiple copies of a target nucleic acid, the method comprising performing multiple sequencing cycles, wherein the sequencing primer in each copy is extended by one nucleotide to identify a complementary nucleotide in a template; after a number of such sequencing cycles, re-phasing the nucleic acid copies; and then resuming the sequencing cycles to identify other nucleotides in the template.

[0041] Rephasing can be performed as many times as necessary to obtain the desired accuracy and signal strength, depending on the error rate of the particular sequencing method that starts with out-of-phase copies. As shown below, rephasing is performed at least once and can be performed 1 to 10 times or 2 to 4 times per sequence read, which can include at least 200, 400, 800 or 1200 sequencing cycles.

[0042] The benefits of re-phasing include extending the number of sequencing cycles with less than a certain percentage of inconsistency, as shown in the working examples below. As an example, as shown, the number of cycles with less than a 2% threshold of inconsistency can be increased by at least 25%, or by at least 1.5 times or 2 times, depending on the initial error rate and the number of re-phasing cycles and events. Re-phasing can extend the number of sequencing cycles with less than 2% inconsistency by at least 100, 200 or 400 cycles or more.

[0043] The present disclosure also provides the reader with kits, reagent combinations, and intermediate mixtures that include any of the reagents and mixtures explicitly or inherently described in the following description, optionally accompanied by instructions for performing rephasing according to the techniques of the present disclosure.

[0044] Other embodiments of the invention are described and illustrated in the following description and accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The use of RNAse HII to cleave the extended strand back to the first incorporated RNA base prior to re-phasing is described. This is one of several "back-off" processes explained below, used in preparation for re-phasing. RNAse HII catalyzes the 5' cleavage of the DNA phosphodiester backbone into strings of nucleotides or ribonucleotides embedded in the dsDNA, leaving behind a 3' OH and a 5' phosphate.

[0046] Figure 2The percentage of DNB templates that were 100% re-phased back to the reference sequence using the dinucleotide method under different re-phasing conditions is shown. Each triplet represents the degree of re-phasing after the first, second or third re-phasing event. All conditions resulted in more than 85% of DNBs being re-phased. There was no re-phasing at all, illustrating that the number of in-phase DNBs was virtually zero.

[0047] Figure 3 The percentage of DNBs that are synchronized is shown. These data illustrate how the percentage of DNBs that are out of phase with only one site is inversely proportional to the percentage of DNBs that are completely in phase. While the percentage of DNBs that are completely synchronized increases between re-phasing events, the percentage of DNBs that are out of phase with only one site decreases.

[0048] Figure 4A and 4B The kinetics of phase inconsistencies for a human reference sequence and a computer-generated random reference sequence are shown, respectively. Under simulated conditions, without the need for re-phasing, inconsistencies rapidly accumulated after 300 sequencing cycles and exceeded 5% at 500 cycles. Two re-phasing events maintained inconsistencies below 2% at 750 cycles. Three re-phasing events maintained inconsistencies below 2% for more than 900 cycles.

[0049] Figure 5 Cumulative cycle shift after the final rephasing event is shown. Negative values ​​correspond to the generation of overlapping sequence regions during rephasing, while positive values ​​correspond to sequencing through the assigned number of cycles. CG duplexes resulted in only about 8% and 40% DNB sequencing beyond the endpoint relative to CA duplexes. This difference is attributed to the higher frequency of CA duplexes in the human genome compared to CG duplexes. DETAILED DESCRIPTION

[0050] The methods disclosed herein can be used to re-phase multiple nucleic acid copies for any purpose. The technology is particularly suitable for nucleic acid sequencing methods, such as array-based massively parallel sequencing using synthesis sequencing.

[0051] Massively parallel sequencing by synthesis is typically performed using DNA arrays, in which cycles of template-directed DNA synthesis are performed on arrays comprising many clonal populations of templates fixed at physically separated positions on a substrate. Examples of clonal populations include, but are not limited to, (i) concatemers having many copies of a template sequence and (ii) clusters of many copies of a linear polynucleotide (e.g., generated using bridged PCR). Sequencing using clonal populations (multiple copies of a template) increases signal strength and mitigates errors that may occur due to unexpected reactions occurring on individual copies.

[0052] I. Terms and Definitions

[0053] In the following description, the following terms are used:

[0054] A "clonal population" refers to one or more template molecules, wherein the clonal population includes many copies (populations) of a sequence corresponding to the same (clonal) target sequence, such that the copies are physically or spatially associated with each other, e.g., contained in or immobilized at discrete locations on a substrate, on separate beads, or in separate compartments (e.g., droplets). The template sequence and target sequence correspond to each other as reverse complements of each other. Thus, the template sequence corresponding to the target sequence may be referred to as the "target sequence complement." In array-based MPS, up to 10 9 Or more spatially separated clonal populations, each with hundreds or thousands of copies of the template sequence, can be distributed on or positioned on a substrate. An example of a clonal population is a DNA nanoball (DNB), which is a single-stranded concatemer with many copies of a target sequence, typically produced by rolling circle amplification. R. Drmanac et al., Science. 327, 78-81, 2010. Another example of a clonal population is an amplicon cluster containing hundreds to thousands of amplicons with the same target sequence, typically produced by bridging amplification (e.g., PCR). In another embodiment, a clonal population is attached to a bead surface (e.g., produced by emulsion PCR; see (Metzker et al., Nat Rev Genet. 11(1): 31-46, 2010). In some methods, a clonal population may contain many copies of a target sequence and its complementary sequence. A clonal population is typically prepared from a "library" such as a genomic or cDNA library.

[0055] "Template", "template sequence", "nucleic acid template", etc. refer to a polynucleotide recognized by a nucleic acid polymerase (e.g., a DNA polymerase). In MPS, a template library is prepared from target molecule DNA fragments (e.g., genomic DNA) connected to an adapter sequence. As known in the art and discussed elsewhere herein, a polymerase catalyzes the extension of a primer hybridized to the template (usually hybridized to an adapter sequence) by the continuous addition of (deoxy)ribonucleotides, wherein each added nucleotide forms a base pair with (i.e., is complementary to) the corresponding base of the template.

[0056] "Target sequence" refers to a nucleic acid sequence (usually a DNA sequence) determined in a sequencing reaction. The target sequence (sometimes referred to as a "reference sequence") is complementary to the corresponding DNA template and is generated by replication.

[0057] "Nucleobase" refers to a nitrogenous base that can base pair with a complementary nitrogenous base of a template nucleic acid. Exemplary nucleobases include adenine, cytosine, guanine, thymine, uracil, inosine, and derivatives thereof. References to thymine herein are to be understood as references to uracil as well, unless otherwise apparent from the context. The terms "nucleobase," "nitrogenous base," and "base" are used interchangeably.

[0058] "Nucleotides" consist of a nucleobase, a sugar, and one or more phosphate groups. They are the monomeric unit of a nucleic acid sequence. In RNA, the sugar is ribose, while in DNA, the sugar is deoxyribose. The nitrogenous bases are derivatives of purines or pyrimidines. The purines are adenine (A) and guanine (G), and the pyrimidines are cytosine (C) and thymine (T) [or in the case of RNA, uracil (U)]. The C1 atom of the deoxyribose is bound to the N1 of a pyrimidine or N9 of a purine. Nucleotides are also phosphates or nucleosides, in which esterification occurs at the hydroxyl group attached to the C5 of the sugar. Nucleotides are typically monophosphates, diphosphates, or triphosphates. "Nucleosides" are similar in structure to nucleotides, but do not include the phosphate unit. Common abbreviations include "dNTP" for deoxynucleotide triphosphates.

[0059] "Nucleic acid" means a polymer of nucleotide monomers. As used herein, the term may refer to single-stranded or double-stranded forms. The monomers constituting nucleic acids and oligonucleotides can specifically bind to natural polynucleotides by means of a regular pattern of monomer-monomer interactions (e.g., base pairing, base stacking, Hoogsteen or reverse Hoogsteen type base pairing, etc.) to form a duplex or triplex form. Non-naturally occurring analogs may include peptide nucleic acids, locked nucleic acids, phosphorothioate nucleoside bonding, bases containing linking groups that allow labeling to attach. When nucleic acids are commonly referred to as "oligonucleotides", their sizes are typically from a few monomeric units to hundreds of thousands or more monomeric units.

[0060] Whenever a nucleic acid (not a template) or an oligonucleotide is represented by a sequence of letters (uppercase or lowercase), such as "ATGCCTG", the nucleotides are in 5' to 3' order from left to right, "A" represents deoxyadenosine, "C" represents deoxycytidine, "G" represents deoxyguanosine, "T" represents thymidine, "I" represents deoxyinosine, and "U" represents uridine, unless otherwise indicated or obvious from the context. As will be understood by those skilled in the art, where the template sequence is shown aligned with the target, the template is the reverse complement of the target and is represented in the 3'→5' direction. See "Scheme A" below. Unless otherwise indicated, the terminology and atom numbering conventions will follow those disclosed in Strachan and Read, Human Molecular Genetics 2 (Wiley Liss, New York, 1999). Typically, nucleic acids comprise natural nucleosides (e.g., deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine for DNA or their ribose counterparts for RNA) linked by phosphodiester bonds; however, they may also comprise non-natural nucleotide analogs, such as modified bases, sugars, or internucleoside linkages. The selection of compositions suitable for oligonucleotides or nucleic acid substrates is guided by texts such as Sambrook et al., Molecular Cloning, 2nd edition (Cold Spring Harbor Laboratory, New York, 1989).

[0061] "Polynucleotide" is used interchangeably with the term "nucleic acid" to refer to DNA, RNA, and hybrid and synthetic nucleic acids, and may be single-stranded or double-stranded. "Oligonucleotides" are short polynucleotides between about 6 and about 300 nucleotides in length. "Complementary polynucleotide" refers to a polynucleotide that is complementary to one strand of a nucleic acid.

[0062] "Reversible terminator" nucleotide is a nucleotide analog that can be incorporated into the growing chain by polymerase, which contains a blocking group (sometimes at the 3'-OH position of deoxyribose). The blocking group prevents the formation of a phosphodiester bond between the nucleotide at the 3' end of the growing chain and the unincorporated nucleotide, and reversibly terminates the further extension of the growing chain. In some cases, the reversible blocking group is a chemical part connected to the 3'-O position of the nucleotide sugar part. The reversible blocking group can be cut by enzymes (such as phosphatases or esterases), chemical reactions, exposure to heat, light, etc., to provide a hydroxyl group at the 3' position of a nucleoside or nucleotide, so that nucleotides can be added by a polymerase. The blocking group prevents polymerization and nucleotides are added to the 3' end of the growing chain. Removing the blocking group allows the polymerization reaction to continue. The reversible terminator nucleotide can be referred to as a "blocked nucleotide", which can be "unblocked" by removing the blocking group. The terms "reversible blocking group", "removable blocking group", "blocking part", "blocking group", "reversible terminator blocking group" etc. are used interchangeably. Unless otherwise indicated in the context, the terms "reversible terminator nucleotide", "reversible terminator", "RT" and "non-labeled reversible terminator (NLRT)" refer to sequencing reagents containing a nucleobase or analog, a deoxyribose or analog, a phosphate and a cleavable (or otherwise removable) blocking group. The reversible terminator can be labeled (e.g., labeled to a fluorescent dye via a cleavable linker) or unlabeled (NLRT; see U.S. Pat. No. 10,851,410). The terms "reversible", "removable" and "cleavable" refer to blocking groups and are used interchangeably. In some cases, a growing chain or oligonucleotide in which the 3'-proto-terminal nucleotide is blocked may be referred to as "blocked", as will be clear from the context.

[0063] As used herein, "unblocked nucleotides" refer to nucleotides that can be incorporated into a growing chain. Unblocked nucleotides can be "natural" or naturally occurring nucleotide monophosphates (N), such as deoxyadenosine monophosphate (A), thymidine monophosphate (T), deoxyguanosine monophosphate (G), deoxycytidine monophosphate (C), deoxyuridine monophosphate (U), or their related triphosphate forms (dATP, dTTP, dUTP, dCTP, dGTP), unblocked analogs, etc. If not otherwise indicated or clear from the context, "nucleotides" refer to naturally occurring nucleotides, nucleotide analogs used in sequencing, blocked nucleotides, or unblocked nucleotides.

[0064] As described herein, a degenerate oligonucleotide used for re-phasing can be used to block the extension of the growing chain, and removal of the oligonucleotide is a type of "unblocking."

[0065] The term "subunit" is sometimes used to refer to a growing chain (or the corresponding portion of a template, such as a monomer in a concatemer) that includes one copy of the target sequence, as will be apparent from the context, and may include associated adapters, primer binding sequences, etc.

[0066] "Substantially all" refers to the proportion of extended primers or growing chains blocked or blocked at the same reference position in a clonal population, meaning more than 90%, preferably more than 95%, 96%, 97%, 98%, 99% or 99.5% (sometimes 100%) are blocked. The proportion of blocked chains can be determined empirically or can be mathematically estimated based on knowledge of the template sequence, the number of re-phasing modules, etc. "Most" and "majority" as used herein refer to 51% or more.

[0067] "Primer" is a kind of oligonucleotide, which can be used as the starting point of nucleic acid synthesis when forming a duplex with a polynucleotide template, and extends along the template from its 3' end, thereby forming an extended duplex. It can be recognized that the complex including the extended primer or growth chain annealed with the template sequence can be referred to as a duplex. The terms "probe", "primer" and "oligonucleotide primer" are interchangeably used in the present disclosure to refer to an oligonucleotide that anneals to the complementary sequence of a nucleic acid template and can be extended by a polymerase by adding nucleotides. The primer adding nucleotides is a "growing chain", "extending duplex" or "extending primer". When dNTP (i.e., nucleoside triphosphate) is added to the 3' end of the primer, pyrophosphate is removed so that nucleoside monophosphate (or nucleotide) is incorporated. Unless otherwise specified, unlabeled or unlabeled reversible terminator nucleotides can refer to (free nucleoside triphosphate or incorporated nucleotide monophosphate) form, which will be clearly seen from the context. Unlabeled or unlabeled reversible terminator nucleotides can be referred to as NLRT. The nucleotide sequence added during the extension process is determined by the sequence of the template polynucleotide. Usually the primer is extended by a DNA polymerase. The primer usually has a length of 9 to 40 nucleotides, or 14 to 36 nucleotides.

[0068] In the context of dinucleotide frequency rephasing (DFR), the terms dinucleotide, "2mer" and "doublet" are used interchangeably. Unless otherwise specified or required, the DFR method can use any combination of two nucleotides, including nucleotide repeat regions, for a total of 16 possible choices.

[0069] The term "sequential sequencing" as used in this disclosure refers to any method of nucleic acid sequencing that includes a cyclic sequence determination of one or more bases at one position in a template nucleic acid, and then the process moves to the adjacent one or more bases until all bases in a particular region of the template have been determined. Synthesis sequencing is exemplary, where single base determination is performed by base-by-base synthesis of a complementary strand in successive cycles, determining the base added in each cycle.

[0070] "Amplicon" refers to the product of a polynucleotide amplification reaction, i.e., a polynucleotide population replicated from one or more starting sequences. Amplicon can be produced by a variety of amplification reactions, including but not limited to polymerase chain reaction (PCR), bridge PCR, linear polymerase reaction, nucleic acid sequence-based amplification, rolling circle amplification (U.S. Patent Nos. 7115400, 4683195; 5210,015; 6174670; 5399,491; 6287824 and 5854033; and U.S. Patent Publication No. 2002 / 0024711).

[0071] "Array" or "microarray" refers to a solid support (or a collection of solid supports, such as beads) having a surface (preferably, but not limited to, a flat or substantially flat surface) that carries a collection of sites containing nucleic acids such that each site of the collection is spatially defined and does not overlap with other sites of the array; that is, the sites are spatially discrete. An array or microarray may also include a non-planar interrogable structure having a surface such as a bead or a well. The oligonucleotides or polynucleotides of the array may be covalently bound to the solid support, or it may be non-covalently bound. Traditional microarray technology is reviewed in, for example, Schena, Ed. (2000), Microarrays: A Practical Approach (IRL Press, Oxford). "Random array" or "random microarray" refers to a microarray in which the identity of the oligonucleotides or polynucleotides is not identifiable, or at least not initially identifiable by their position, but can be determined by specific biochemical detection techniques performed on the array. See U.S. Patent Nos. 6,396,995; 6,544,732; 6,401,267; and 7,070,927; PCT Publications WO 2002 / 073504 and 2002 / 082098; and U.S. Patent Publication Nos. 2002 / 0207482 and 2002 / 0087362.

[0072] "Solid support" and "support" refer to a material or group of materials having a rigid or semi-rigid surface. A microarray typically includes at least one planar solid support, such as a microscope slide.

[0073] "Incorporation" means becoming part of a nucleic acid molecule. In SBS, when the polymerase is added to the DNA strand by the 3' position of the pentose sugar of one nucleotide (ie, the 3' nucleotide on the DNA strand) and the 5' position of the pentose sugar on the adjacent nucleotide, ie, RT is added to the DNA strand.

[0074] In the context of labeled affinity reagents, "label" means any atom or molecule that can be used to provide a detectable and / or quantifiable signal. Suitable labels include radioisotopes, fluorophores, chromophores, mass labels, electron-dense particles, magnetic particles, spin labels, molecules that emit chemiluminescence, electrochemically active molecules, enzymes, cofactors, and enzyme substrates.

[0075] "Restoring phase," "resetting phase," and "re-phasing" are used interchangeably in this disclosure.

[0076] "Repositioning" or "backtracking" is a process used in conjunction with rephasing to backtrack the sequencing position 5 to 50 bases upstream of the growing chain. As described below, this provides the user with an overlap of sequencing information obtained before and after the rephasing event, rather than creating a gap.

[0077] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polymerase" refers to one reagent or a mixture of reagents, and reference to "the method" includes reference to equivalent steps and / or methods known to those skilled in the art.

[0078] Unless otherwise stated or required, other technical and scientific terms used in this disclosure have their common meanings.

[0079] Where a range of values ​​is provided, unless the context clearly dictates otherwise, each intervening value to one tenth of the unit of the lower limit between the upper and lower limits of the range and any other stated or intermediate values ​​within the stated range are encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges and are also encompassed within the present invention, subject to any specifically excluded limits within the stated ranges. Where the stated range includes one or two limits, ranges excluding one or both of these included limits are also encompassed within the present invention.

[0080] Unless otherwise indicated, the practice of the present invention may employ conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant technology), cell biology, biochemistry and immunology, which are within the technical scope of the art. These conventional techniques include polymer array synthesis, hybridization, connection (ligation) and hybridization detection using labels. Specific descriptions of suitable techniques may be obtained by reference to the examples below. However, other equivalent conventional procedures may certainly be used. These routine techniques and descriptions can be found in standard laboratory manuals, for example, Genome Analysis: A Laboratory Manual Series (Vols. I-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 Ed.) Freeman, NY, Gait, "Oligonucleotide Synthesis: A Practical Approach" 1984, IRL Press, London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3rd Ed., WH Freeman Pub., New York, NY, and Berg et al., (2002) Biochemistry, 5th Ed., WH Freeman Pub., New York, NY.

[0081] ii. Synthesis sequencing using reversible terminators

[0082] The technology of the present invention is generally applicable to sequencing methods, such as synthetic sequencing (SBS) using reversible terminators. In these methods, each sequencing cycle (usually) identifies one nucleotide. Various SBS methods are known. See, for example, R. Drmanac et al., Science. 327, 78–81, 2010; PCT Patent Publication WO 2016 / 133764; Mardis E., 2017, Nature Protocols Nature Protocols 12: 213–218; Margulies et al., 2005, Nature 437: 376-380; Ronaghi et al., 1996, Anal. Biochem. 242: 84-89; Constans, A, 2003, The Scientist 17 (13): 36; and Bentley et al., 2008, Nature 456 (7218): 53–59. Determining the sequence of a nucleic acid generally requires multiple cycles of reactions that generate signals corresponding to the identity of one or more nucleotides in the sequence. In one approach, this is accomplished using primer extension reactions on a clonal population containing many copies of the sequence to be determined. In some approaches, the dNTP analogs (reversible terminators) incorporated by primer extension are labeled with fluorescent dyes. In some approaches, the incorporated dNTP analogs are not linked to dyes and are detected by affinity reagents (e.g., antibody sequencing). See U.S. Pat. No. 10,851,410, describing Sequencing arrays comprising a large number (usually hundreds of millions) of positions are commonly used and are typically included in flow cells designed for automatic sequencing devices. The array can be patterned, or the site of template attachment can be an arbitrary position on the substrate. DNA sequencers that perform sequencing by synthesis are commercially available, such as the BGISEQ-500 from BGI (Shenzhen, China) and the NextSeq from Illumina Inc. (San Diego, California, USA). Some SBS methods include detecting protons released when nucleotides are incorporated into extension products. For example, sequencing based on detection of released protons can use electrical detectors and related technologies commercially available from Ion Torrent (Guilford, Conn., A Life Technologies affiliate), or sequencing methods and systems described in U.S. Patent Publications No. 2002 / 0026082A1; No. 2002 / 0127589A1; No. 2010 / 0137143A1; or No. 2010 / 0282617A1.

[0083] III. Phase

[0084] In a sequencing reaction applied to a clonal population, the growing chain is extended in each cycle, typically by adding one nucleotide per sequencing cycle. When two growing chains in a clonal population contain the same number of incorporated nucleotides (e.g., corresponding to the number of incorporation cycles of the sequencing reaction) and terminate at the same position of the corresponding template sequence, the two growing chains are "in phase" in any sequencing cycle. Growing chains that are "in phase" have the same base sequence at the 3-primitive end.

[0085] IV. Phase Loss / Inconsistency

[0086] The sequencing of clonal colonies provides advantages, such as stronger signals, improves accuracy, and in some methods, reduces the amplification errors that occur in the process of producing clonal colonies. However, such sequencing requires the coordinated extension of templates in each clonal colony. That is to say, all or most of the growing chains in the clonal colony must be extended in each cycle, and each cycle extends the same number of nucleotides (usually one nucleotide). However, in fact, nucleotides are incorporated into many growing chains at any given position on the array and can be "out of phase". This refers to the fact that in any given sequencing cycle in which nucleotides are incorporated into the growing chain in the clonal colony, there may be heterogeneity in sequencing chemistry, resulting in some growing chains that are not incorporated with nucleotides and / or some growing chains that are incorporated with more than one nucleotide. For example, for each continuous nucleotide, the frequency of sequence reaction is very small and can not enter the next sequencing cycle in a normal manner. Unfinished sequencing cycles can return to the same intermediate product produced by the previous cycle. In this case, the next cycle will identify the nucleotides that should be identified in the earlier cycle. This specific copy of DNA will be a cycle phase (lag sequencing) after other copies. Alternatively, a sequencing cycle that skips one nucleotide or processes two nucleotides in the same cycle will precede the other copies by one cycle (run-on sequencing). Phase loss (or reduction) can be referred to as an increase in inconsistency.

[0087] The term "inconsistency" is commonly used in the field of sequences, as in sequencing, to indicate that a base does not match the known reference identity of the base at that position in the read. Inconsistency as used herein refers to the proportion of chains that are out of phase in a clonal population. Percent inconsistency refers to the percentage of calls at positions that do not match a reference sequence, and is an indicator of the proportion of out-of-phase growing chains (as more primers become out of phase, the probability of producing inconsistent calls increases). If the mixing of intensities does not reach a state where the position is "flipped" to an erroneous call, then if only some growing chains are out of phase, the call may not be inconsistent. However, even if it is not reflected in the mis-call reads, reducing the number of out-of-phase chains also increases signal strength, allowing longer reads and having other benefits.

[0088] The result of each copy of the amplified DNA (e.g., target sequence) being out of phase is that in subsequent sequencing reaction cycles, the information obtained from that DNA copy or target sequence will be incorrect. Therefore, as sequencing continues, the cycle sequencing process will accumulate more and more out-of-phase copies, and the proportion of correct in-phase copies will decrease over time.

[0089] Depending on the sequencing method and the reagents used, we have found that after 200 cycles, typically about 20% of the template copies may be lagging behind, while about 10% are ahead of phase. This will have the effect of reducing the signal-to-noise ratio, blurring the readout, until the information is no longer sufficiently accurate. This effectively limits the length of sequence reads that can be obtained. The technology of the present invention provides the user with the opportunity to re-phase the DNA amplicons between sequencing cycles.

[0090] "Schemes" are used in this disclosure, such as "Scheme A" below, to describe a population of clones in which the individual growing chains (S1-4) are in phase or out of phase. A "target sequence" is sometimes referred to as a "reference sequence". The target sequence is complementary to the template sequence. Importantly, the template and target sequences do not necessarily (and usually do not) represent all of the template or target nucleotides in the clone or growing chain (extension primer). For example, in Scheme A below, the 5'-most C of the sequence shown (cgta...) can be the 50th, 100th, 200th, etc. nucleotide in the extension primer (where the first incorporated nucleotide of the extension primer is considered to be nucleotide 1). For clarity and convenience, for example, we adopt the convention of referring to the 5'-most nucleotide (C) as the nucleotide incorporated in the first sequencing cycle, rather than the 200th.

[0091] Plan A

[0092]

[0093] In scheme A, chains S2 and S3 show the sequence of the growing chains in phase after 15 sequencing cycles, where the nucleotide incorporated in the next cycle will be A. S1 shows the sequence of the growing chain where an additional nucleotide is incorporated, and where the nucleotide incorporated in the 16th cycle will be C (i.e., running sequencing). S4 shows the sequence of the growing chain where no nucleotide is incorporated in the 15th cycle, where the nucleotide incorporated in the 16th cycle will be T (i.e., lagging sequencing).

[0094] Scheme A The following example shows the correct or reference sequence for the 3' region of a fully extended growing chain, and four examples show the sequence of the last 15 extensions (15 sequencing cycles) of a growing chain of four subunits. Individual growing chains are sometimes referred to as "subunits". It will be understood that the following description refers to one clonal population (e.g., one "spot" on an array), and the re-phasing steps described herein occur on arrays of thousands, millions, or hundreds of millions of clonal populations with different sequences.

[0095] In this illustration, some subunits may terminate at T. S2 and S3 reflect the accurate incorporation of 15 nucleotides in 15 sequencing cycles. In this example, one subunit, S4, lags by one base and incorporates G in this cycle. Subunit S1 runs-on relative to S2 and S3 and terminates with A.

[0096] V. Re-phasing; phase restoration

[0097] The present disclosure provides methods and reagents for recovering phase in step-by-step sequencing of cloned DNA using reversible chain terminators (RT). For high-quality long sequence reads longer than 300 or 500 bases, it is necessary to remove the lag (usually -1 or -2 bases) and run (usually +1 base) sequencing copies that have accumulated (especially after 100 or more sequencing cycles). Re-phasing the growing chain at the site on the array will improve sequence quality and read length. Re-phasing when 20% or more, 30%, or greater than 50%, or greater than 60%, or preferably greater than 70%, or 80% of the DNA clones are sequenced on the array will provide sufficient yield for long MPS reads (500 bases to 700 bases or 600 bases to 1000 bases or even longer). In this article, the re-phasing percentage refers to the percentage of clonal structures, in which each extended chain of the subunit of the structure has been reset to the same sequence length and has terminated at the same position in the target fragment representing the clonal structure (e.g., DNB).

[0098] VI. Method 1: Rephasing single nucleotide frequencies with forward runs using blocker-1

[0099] The re-phasing method 1 includes the incorporation of 3 natural nucleotides (e.g., A, C, G) plus a 3' reversibly blocked nucleotide (e.g., T). Scheme A shows a heterophasic population of terminal blocking group cleavage at the end of a standard sequencing cycle (as described above). Subunit s1 runs relative to s2 and s3 and terminates with A. In this article, "subunit" can refer to but is not limited to a monomer in a concatemer or a single template polynucleotide in a cluster. Some subunits can terminate at T, and in this example, one subunit lags one base and G is incorporated in this cycle.

[0100] Plan A

[0101] Clone Group 1

[0102]

[0103] In this method, the extended chain is run forward with a mixture of 3 native (i.e., unblocked) and 1 blocked nucleotides. In this example, each subunit will terminate at a T. However, some subunits will terminate at a T at a different position in the sequence, so the desired effect of all subunits being in phase will not be achieved. This is because T is the terminating base in some subunits.

[0104] To "reset the phase", a mixture of 3 natural and 1 blocked nucleotides is incorporated, for example, using a polymerase and a nucleotide mixture having 1 blocked nucleotide and 3 unblocked (e.g., natural) nucleotides, and the growing chain is run forward. The blocked nucleotide can be any of the 4 nucleotides: in this example, T is blocked (denoted as "T*"). Extension or "running" of the Scheme A population results in the Scheme B population shown below. The incorporated bases are shown in underlined, uppercase bold font.

[0105] Plan B

[0106] Clonal population 1 after running with blocked T (T*) and 3 natural nucleotides

[0107]

[0108] In this embodiment, only T is blocked. The growing chain in each clonal population on the array will be extended until A appears in the template, at which point the incorporation of T will terminate the extension of the single strand (i.e., all subunits terminate at T because T is the only nucleotide with a 3' blocking group). Although in Scheme A, two of the four subunits (s1 and s4) are out of phase, after re-phasing in Scheme B, only one of the four subunits is out of phase, and the inconsistency is reduced. Subunit S1 is now in phase with respect to s2 and s3. Subunit s4 is now further out of phase because the initial phasing spans the "T" as the terminating nucleotide.

[0109] It should be appreciated that in this example, no sequence information is available for positions 16, 17 and 18 (ACG). Such gaps in information can be addressed in a variety of ways, as discussed below.

[0110] Protocol C illustrates the effect of re-phasing the same Protocol A clonal population (ie, same target sequence), but using C as the blocking nucleotide.

[0111] Plan C

[0112] Clonal population 1 after running with blocked C (C*) and 3 natural nucleotides

[0113]

[0114] In this example, only C is blocked, and the growing chains in each clonal population will be extended until G appears in the template, at which point incorporation of C* will terminate the extension of each chain. While two of the four subunits (s1 and s4) are out of phase in scheme A, all s1-s4 are in phase in scheme C, reducing inconsistency.

[0115] Scheme D illustrates the extension of a different clonal population, "clonal population 2" (having a target sequence different from that of Scheme A). For Scheme C, T is used as a blocking nucleotide in the re-phasing step. In Scheme D, two of the four strands are in phase.

[0116] Plan D

[0117] Clonal population 2 followed up with closed T

[0118]

[0119]

[0120] In this re-phasing method, in each re-phasing event, one nucleotide is blocked and three nucleotides are not blocked. Although any one of the four nucleotides can be selected as blocked, the efficiency of the method can vary based on the nucleotide composition of the target sequence (for example, such as the difference between the mammalian genome sequence and the bacterial genome sequence of the high GC library compared to the low GC library). The re-phasing results of any given clonal population depend on the template sequence and the blocked nucleotides selected in the "recovery" cycle. However, in arrays with a large number of populations, these are usually net gains in phase. Typically, the ability of method 1 to correctly re-phase the chain is limited by the frequency of blocked nucleotides (T or C in the above embodiment) in the target sequence.

[0121] In order to increase the proportion of out-of-phase chains that return to phase, or to achieve a higher probability of returning all subunits to phase, the frequency of termination features in the target sequence or template should be less common. This is achieved by stopping the forward running step at a selected dinucleotide rather than at a single nucleotide in the template. Methods 2-4 described below use this approach.

[0122] VII. Method 2: Dinucleotide Frequency Rephasing (DFR) with Forward Run

[0123] In this method, each growing chain is extended until the position in the target sequence corresponding to the selected dinucleotide sequence. The dinucleotide sequence may also be referred to as a "duplex" or "2mer". Any dinucleotide selected from 16 possible combinations may be used. For the purposes of the following description, the dinucleotide TC is identified. In this description, the clonal population is represented as Scheme E, as shown below.

[0124] Plan E

[0125]

[0126] In one embodiment, the steps of the DFR cycle are as follows. One of ordinary skill will recognize that the steps described here are illustrative, but specific reagents or methods may be varied when implementing the method.

[0127] Step 1. Extend the growing chain of the clonal population by incorporating four nucleotides, one of which (in this example, T) is blocked, and three of which (in this example, A, C, and G) are not blocked. The blocked nucleotide corresponds to the first nucleotide of the selected duplex. The blocked first nucleotide can be blocked by a group called "blocking group-1", which can be represented by an asterisk (e.g., T*).

[0128] Plan F

[0129] Cloning population 1 dinucleotide (TC) method to block T

[0130]

[0131] Step 2. Removal of polymerase and excess (unincorporated) nucleotides. It will be appreciated that removal can be accomplished using any suitable method and can generally be referred to as "washing to remove excess nucleotides and polymerase." In one approach, "washing" is accomplished by flowing a buffer through a flow cell containing the array.

[0132] Step 3. The terminal group-1 blocking group is unblocked with an unblocking agent, returning the 3' group to the hydroxyl moiety capable of accepting further nucleotide extension. Thus, each subunit terminates at a T base, as shown in Scheme G. Methods for removing blocking groups or otherwise unblocking reversible terminators are known in the art and are discussed below.

[0133] Plan G

[0134]

[0135] Step 4. An incorporation step is performed by adding a reversibly terminated nucleotide with blocking group-2. The blocked nucleotide corresponds to the second base of the selected dinucleotide (C in this example). This step "reads" the next consecutive base to see if it corresponds to (or correctly identifies) the second nucleotide (C) of the selected dinucleotide (TC). The blocked nucleotide corresponding to the second nucleotide of the dinucleotide has a different blocking group ("blocking group 2") than blocking group-1. Blocking group-2 can be indicated by a reverse arrow (e.g., ) indicates. In addition to incorporating the nucleotide corresponding to the second base of the dinucleotide blocked with blocking group 2 (C in this example), nucleotides corresponding to the other three nucleotides blocked with blocking group-1 (e.g., A, G, T) are also incorporated to advance all chains by one base. By adding all four nucleotides, the possibility of C mismatch is advantageously reduced when the template is not G.

[0136] According to the method, under the same conditions as the removal of blocking group-1, blocking group-2 is not unblocked. For example, in some cases, different reagents are used to remove group 1 and group 2 blocking groups, and blocking group 2 cannot be unblocked with the same reagent that can unblock group 1. In other words, it is possible to select to remove blocking group-1 without removing the conditions of blocking group-2. For example, in one method, block 1 (Block-1) is-O-azidomethyl, which can be removed by phosphine (e.g., TCEP) cutting, and block 2 is-O-NH2, which can be removed by sodium nitrite cutting. See Hutter et al., 2010, Nucleosides Nucleotides Nucleic Acids.29 (11) doi: 10.1080 / 15257770.2010.536191. Many other groups are available, including those containing disulfide bonds. See discussion below. The method can be carried out using a reversible terminator and a reagent that partially or completely unblocks blocking group 1 with a block-2 unblocking condition. It will be appreciated that a plurality of different blocking groups may be used (eg, two different blocking groups may replace blocking group 1 and / or two different blocking groups may replace blocking group 2 if the relationship relative to the deblocking conditions is maintained).

[0137] In some methods, the same DNA polymerase is used for all steps in the sequencing and re-phasing process. For example, in some cases, a polymerase that can recognize and incorporate nucleotide analogs with blocking group-1 and incorporate nucleotide analogs and / or natural nucleotides with blocking group-2 is used. Alternatively, a mixture of polymerases with different properties can be used and / or different polymerases with different properties can be used in different steps, rather than in the same mixture. In some cases, the incorporation step can be performed for 30 seconds to 2 minutes; however, this can vary with the choice of polymerase and other reagents and can be optimized by professionals.

[0138] Scheme H, shown below, shows that when applied to the illustrative clonal population of Scheme G, step 4 does not result in the incorporation of "C" into any of the growing chains S1-4.

[0139] Scheme H

[0140]

[0141]

[0142] Step 5. Repeat step 2 ("Washing").

[0143] Steps 1-5 may be referred to as "re-phasing rounds". The re-phasing round steps may be repeated multiple times to reduce the overall inconsistency of numerous different clonal populations on an array or other system. The other three rounds are described below. A "re-phasing module" or "re-phasing event" (i.e., all steps taken to achieve substantially complete re-phasing at a specified point in the sequencing process) typically consists of 5 to 15 re-phasing rounds (e.g., steps 1-5 repeated approximately 5 to 15 times). Preferably, the re-phasing module includes less than 10 re-phasing rounds (e.g., 4-9, 5-7, 5-9, 6-9, 7-9, 8-9 or 9 rounds). In some cases, 10 or 11 re-phasing rounds are performed.

[0144] The final step in the rephasing event is the unblocking of block 2. The unblocking of block 2 occurs only once in the rephasing module.

[0145] Round 2

[0146] Repeat step 1 (Scheme I):

[0147]

[0148] Repeat steps 2 and 3 (Scheme J):

[0149]

[0150] Repeat steps 4 and 5 (Scheme K). It is not incorporated into any chains during this step.

[0151]

[0152] Round 3

[0153] Repeat step 1A (Scheme L):

[0154]

[0155] Repeat steps 2 and 3 (option M):

[0156]

[0157] Repeat steps 4 and 5 (add )(Scheme N):

[0158]

[0159] Round 4

[0160] Repeat step 1 (option O):

[0161]

[0162]

[0163] Repeat steps 2 and 3 (option P):

[0164]

[0165] Repeat steps 4 and 5 (option Q):

[0166]

[0167] Unblocking steps

[0168] Block-2 Unblocking (Scheme R):

[0169]

[0170] After removal of block 2, conventional sequencing can be resumed.

[0171] VIII. Method 3: Dinucleotide Frequency Rephasing (DFR) with Forward Run

[0172] Method 3 is similar to method 2, except that in step 4, the nucleotide corresponding to the second base (C in this example) of the dinucleotide blocked with blocking group 2 is added, but the nucleotides corresponding to the other three nucleotides (e.g., A, G, T) blocked with blocking group 1 are deleted.

[0173] IX. Method 4: Dinucleotide Frequency Rephasing (DFR) using Block 1 and Oligonucleotide Block Forward Run

[0174] In this method, the re-phasing position is still a dinucleotide. However, instead of using block 2 to identify the second nucleotide in the dinucleotide, a degenerate oligonucleotide is used to identify the second nucleotide. The steps of the DFR cycle with oligonucleotide blocks are as follows. For the purpose of illustration, we assume that the starting configuration of the clonal population is as shown in Scheme E above.

[0175] Step 1. Incorporate three natural and one reversibly blocked nucleotide with blocking group-1. As an example, the T nucleotide can be a nucleotide containing blocking group-1, and A, C and G are unblocked (natural nucleotides). The result can be represented as Scheme F, as described above.

[0176] Step 2. Washing to remove polymerase and excess (unincorporated) nucleotides.

[0177] Step 3. Removal of blocking group 1 from the terminal nucleotide yields a 3'OH group that is able to accept further nucleotide extension. The result can be represented as Scheme G, as described above.

[0178] Plan G

[0179]

[0180] Step 4. Add the 5' phosphorylated oligonucleotide and a ligase (eg, T4 DNA ligase). Other suitable ligases include, but are not limited to, T3 DNA ligase, T7 DNA ligase, Taq DNA ligase.

[0181] 5'-phosphorylated oligonucleotides usually have a limited base at the 5' position, a degenerate nucleotide composition and a 3' block. "Degenerate oligonucleotides" used in this article can be an oligonucleotide pool, in which multiple bases (sometimes all four bases) occupy many or each position in the oligonucleotide (except the fixed nucleotides usually at the 5' end). Alternatively, or additionally, degenerate bases and / or universal bases can be used at many or each position. The example of universal bases includes 5' nitroindole and deoxyinosine. Alternatively, the 5' end of the oligonucleotide can be pre-adenylated so that the ligase connects the oligonucleotide to the 3' hydroxyl of the termination chain. 5' phosphorylation can also be achieved by co-reacting with T4 polynucleotide kinase during the ligation reaction to add 5' phosphate groups to unphosphorylated oligonucleotides. As pointed out, degenerate oligonucleotides are sometimes "partially degenerate", that is, some positions can be fixed, as long as this does not affect the function of the oligonucleotide (for example, annealing with different sequences at the position on the template).

[0182] As noted, the nucleotide at the 5' position of the oligonucleotide is fixed and corresponds to the second nucleotide of the selected dinucleotide. If both the 5' nucleotide and the second nucleotide of the oligonucleotide can form base pairs with the same base in the template, the nucleotide at the 5' position of the oligonucleotide "corresponds" to the second nucleotide of the selected dinucleotide. For example, if the second nucleotide is A, T, G or C respectively, the 5' nucleotide can be A, T, G or C respectively. In a preferred embodiment, the second nucleotide is T, and the 5' nucleotide is uracil. That is, in a method, the 5' nucleotide of the oligonucleotide can be a uracil base, so that it will be paired with the A base in the template (such as DNB), and the A in the template corresponds to the T in the target (reference) sequence. The length of the oligonucleotide is generally 6 to 12 bases or longer. In some embodiments, the length is 8, 9, 10, 11 or 12 bases (including 5' fixed bases).

[0183] For example, an oligonucleotide having the following general structure may be used:

[0184] 5'-phos-U(N) Z -X["Degenerate Oligonucleotide #1"]

[0185] wherein "5'-phos" is a phosphorylated nucleotide, U is uracil, X is a blocking structure (i.e., a structure that prevents polymerase-mediated extension of the oligonucleotide), which may be an irreversible blocking structure, Z is 6-20, preferably 6-15, more preferably 6-12, and (N) Zis a degenerate oligonucleotide sequence. In certain embodiments, Z is 9. Examples of irreversible closed structures are dideoxynucleotides and reverse bases (3'-3' bonds, provided by oligonucleotide manufacturers, such as Integrated DNA Technologies, Coralville, Iowa). As the length of the oligonucleotide increases, the probability of mismatching sequences with the template at the 3' end of the oligonucleotide increases. This itself can inhibit polymerase extension from the 3' end of the closed oligonucleotide.

[0186] Although a construct is used as an example, in which uracil is the 5'-terminal nucleotide in the oligonucleotide and is paired with the T base in the template, in principle, cleavage can also be performed by other mechanisms, including but not limited to the incorporation of ribonucleotides and synthetic nucleotides to generate a cleavable site. The method can be performed using any 5'-base, wherein the 5'-base corresponds to a second nucleotide that causes the double-stranded molecule to be cleaved 5' of the 5'-base, for example, when the 5' nucleotide is the cleavage site, such as a removable or abasic nucleotide. In one embodiment, the oligonucleotide is:

[0187] 5'-phos-B(N) Z -X["Degenerate Oligonucleotide #2"]

[0188] Wherein B defines a cleavable site. In some cases, B is a removable or abasic nucleotide. In some cases, B is an A ribonucleotide.

[0189] In other embodiments, the cleavable base position of the oligonucleotide need not be located at the first position of the oligonucleotide. For example, the cleavage at the second position of the oligonucleotide can also be performed with a degenerate base at the 5'-position. However, in this embodiment, the first base of the oligonucleotide will become the terminal base of the growing chain, but its identity is unknown from the continuous sequencing process.

[0190] Using degenerate oligonucleotide #1, if the last base added in the polymerase forward run step is T, as described in the illustrative examples discussed above (see, e.g., Scheme F above and Scheme AA below), the dinucleotide recognition sequence will be the TT dinucleotide in the target sequence. The first base (T) is determined by the run step, and the second base (T) corresponds to the 5' nucleotide of the oligonucleotide (U).

[0191] Plan AA

[0192]

[0193] As shown below (Scheme BB), if T is present in the target sequence after polymerase termination (S1-3), then degenerate oligonucleotide #1 binds and blocks extension. If T is not present in the target sequence at the position after polymerase termination (S4), then degenerate oligonucleotide #1 will not bind and extension can continue in the next cycle.

[0194] Scheme BB

[0195]

[0196] In this method, the re-phasing module includes steps 1 to 5 repeated approximately 10 times, followed by cleavage of the uracil base. The properties of the subunits after 3 rounds of extension and ligation are shown below (starting from Scheme E)

[0197] In each round of extension and ligation, if the T is followed by a non-T base, each strand will continue to extend and terminate at T. If the following base is also a T, then ligation of the 9-mer oligomer will proceed efficiently and effectively terminate any further extension.

[0198] Shown below are the properties of the subunits after 4 rounds of extension and ligation.

[0199] Solution CC

[0200]

[0201] In order to restart sequencing with more number of homogeneous subunits, the extended and oligomeric closed chains are treated with an enzyme mixture of uracil-DNA glycosylase (UDG) and apurinic / apyrimidinic endonuclease 1 (Ape1) or similar abasic site endonucleases. This cuts and removes the uracil base, leaving a T-terminal base with 3'-OH to continue sequencing. For all DNBs, the first sequencing base will be the second T of the TT pair. Other examples of introducing cleavable sites include introducing a single ribonucleotide core base into the oligonucleotide so that after the oligonucleotides are connected, the single ribonucleotide core base is surrounded by DNA bases. RNaseHII nuclease, an enzyme specific to this sequence type, can then be used to excise RNA bases, subsequently lose the 3' side DNA sequence, and leave the 3' DNA end for further extended sequencing. For example, in scheme CC, instead of the uracil base in the second position, the RNA base is incorporated by being located at the 5' end of the connecting oligomer, and then cut by RNaseHII. The T base that was the last position of the polymerase extension is now the terminal position of the continuous chain sequencing extension again. Other methods of incorporating cleavable bonds in oligonucleotides include the use of phosphorothioate bonds or "bridged sulfur" bonds with silver nitrate cleavage (reference PMID: 2027751 Ma et al., Nucleic Acids Res. 1991 Apr11; 19 (7): 1437-41.).

[0202] X. Fallback and other methods for re-adjusting the start site

[0203] Before implementing the above-described re-phasing method, the user may wish to realign the starting point of the strand synthesized in the sequencing reaction by 5 to 50 bases backward or upstream (" Fallback ”). The reason for this is that in its simplest form, there is no sequence determination during rephasing. Backtracking or repositioning the strands being synthesized prevents rephasing from leaving gaps in the sequence, and instead presents the user with regions of sequence overlap to ensure continuity. There are several ways to achieve this, as described in the following sections. The backtracking process itself can aid the rephasing process, but can be limited, by being restricted to single nucleotides, the probability of certain subunits being out of phase is greater than if it were a less frequent sequence event.

[0204] A. Realignment of the start site of the forward running cycle by incorporation of uracil

[0205] A fallback method is to incorporate uracil into the chain synthesized during the sequencing reaction and then cut with an enzyme at the uracil. In this method, before starting the phase reset module, about 20 cycles (e.g., 5 to 30 or 10 to 25 cycles), uracil with a reversible terminator is incorporated to replace the reversible terminator T. Sequencing continues for another 20 to 30 cycles or 20 to 50 cycles, and then the uracil site is cut with an enzyme mixture of uracil-DNA glycosylase (UDG) and apurinic / apyrimidinic endonuclease 1 (Ape1) or similar abasic site endonuclease.

[0206] The following diagram (Scheme HH) shows the first incorporation step of the uracil reversible terminator. Some subunits will fail to incorporate uracil because there is no "A" in the template (eg, DNB) at that particular cycle of the template or clone.

[0207] Scheme HH

[0208]

[0209] Uracil was incorporated for 10 to 20 cycles during sequencing to ensure that most DNBs had incorporated uracil in most subunits.

[0210] Solution II

[0211]

[0212] After uracil cleavage, some subunits will be in phase, while others will be out of phase.

[0213] Plan JJ

[0214]

[0215] After dinucleotide frequency re-phasing (DFR) using blocks 1 and 2 run forward, the amplicons were configured as follows:

[0216] Solution KK

[0217]

[0218] B. Readjustment of the start site of the forward running cycle using phosphorothioate nucleotides

[0219] This method of generating sequence back-off uses an exonuclease in combination with modified nucleotides that block exonuclease digestion beyond a specified region of the extending sequencing strand.

[0220] The first stage of the process is to perform standard sequencing until a predetermined number of cycles. For example, at cycle 200 of the sequencing run, the nucleotide incorporation mixture of the reversibly terminated nucleotide is switched to a nucleotide incorporation mixture containing a 5'-α-phosphothioate-modified nucleotide such as 2'-deoxythymidine-(α-thio)-triphosphate. The phosphorothioate bond replaces the non-bridging oxygen on the α-position phosphate of the triphosphate moiety, but the nucleotide also has a reversible terminator blocking group for the sequencing method. All four bases A, C, G and T will be modified by (α-thio)-triphosphate.

[0221] Sequencing with (α-thio)-triphosphate nucleotides can last for about 5 or more cycles, ideally at least 6 cycles. Since mixed isomers of (α-thio)-triphosphate nucleotides are feasible, the ability of the thioate group to block nucleases can be limited to one of the isomeric forms. See Yang Z. et al., 2007, Nucleic Acids Res. 35, 3118-3127. By incorporating (α-thio)-triphosphate nucleotides at multiple positions, a high percentage of chains that are paused during exonuclease truncation of thiophosphate-modified nucleobases is ensured. If a pure preparation of an isomeric form that can produce nuclease resistance can be guaranteed, fewer incorporation cycles will be required.

[0222]

[0223] 2′-deoxythymidine-(α-thio)-triphosphate

[0224] After at least about 6 incorporation cycles (6-8 cycles), the sequencing mixture is then switched back to the standard 5' triphosphate nucleotides. Then, before the backoff process begins, sequencing is continued for 30 cycles (30-50 cycles). A DNA exonuclease (e.g., exonuclease III) that is specific for the 3' digestion of the recessed strand of the double-stranded structure is then applied to the DNB array to produce a controlled exonuclease reaction, thereby continuously degrading the primer strand from the 3' end. See Rogers S. et al., 1985, Methods Enzymol.65, 201-211. Once the exonuclease process reaches the phosphorothioate bond, the exonuclease reaction is blocked and the backoff process terminates.

[0225] The timing and reaction conditions are selected to ensure that the exonuclease reaction does not overshoot the required 30 base backoff. This minimizes any undesirable side reactions, such as the reported ability of exonuclease III to digest both single-stranded and double-stranded DNA strands. Initiation of the re-phasing process can now begin, which allows forward running within the 30 base window to ensure that sequence coverage of the target fragment is not lost.

[0226] C. Use nickase to realign the start site of the forward running cycle

[0227] The start site of the forward running cycle can also be readjusted using a nickase. The restriction endonuclease Nt.CviPII is a nickase that recognizes the sequence CCd (where D represents A, G, or T instead of C) on double-stranded DNA, and cleavage occurs only on one strand of the duplex. Nt.CviPII will cleave to the 5' side of the dinucleotide CC on the target DNA.

[0228] To target CC dinucleotides near the ends of DNA strands generated during the polymerase-based sequencing process, rather than other CC dinucleotide sequences throughout the duplex read (sequencing-generated strand) and template (e.g., DNB) strand, the enzyme needs to target a local region of the ends, e.g., 20-40 bases. This can be accomplished by using Nt.CviPII enzymes and a suitable Sequencing is achieved by generating fusion proteins between antibodies. The antibody recognizes the terminal incorporated bases through the 3' blocking group and base type. The fusion of Nt.CviPII enzyme and antibody will produce a locally constrained enzyme at the 3' end of the extended chain. Only CCD nucleotides in the extended chain that are in close proximity to the 3' end (close to 15 or 20 or 25 or 30 or 40 bases) will be targeted.

[0229] The DNB strand can be protected from targeting by incorporating a thiolation bond during the synthesis of the DNB strand or by other methods (e.g., restriction enzyme-antibody fusions). If both the DNB and sequencing strand are thiolated or otherwise modified—except in the last 15 to 40 bases of the DNA strand generated by sequencing before the rephasing cycle—free nicking enzyme in solution can be used.

[0230] To restart sequencing with a greater number of in-phase subunits, the extended and blocked 2 subunits are cleaved to remove the blocking group, leaving a C-blocked base with a 3′-OH to continue sequencing. For all DNBs, the first sequenced base will be after the C of the TC pair.

[0231] D. Other methods for re-adjusting the start site

[0232] RNA bases can be used as alternatives to uracil incorporation. It is likely that polymerases containing certain mutations can incorporate RNA bases in addition to accepting reversible terminators. See Gardner et al., 2019, Front Mol Biosci. 2019;6:28. However, the A485L mutation reduces discrimination of rNTPs and allows incorporation of up to 20 ribonucleotides.

[0233] Digestion with RNAse HII will then allow the extended chain to be cleaved back to the first incorporated RNA base. RNAse HII can remove the RNA-containing fragments, leaving a 3'-OH group on the remaining DNB hybridized chain that can continue to extend. The RNA base also allows all four nucleotide bases to be used for incorporation and cleavage. This can be done as Figure 1 Instructions shown.

[0234] DNA endonucleases catalyze the 5' cleavage of the DNA phosphodiester backbone into ribonucleotides or strings of ribonucleotides embedded in the dsDNA, leaving behind a 3' OH and a 5' phosphate.

[0235] Another method to remove 15 to 30 bases from the sequencing strand prior to forward rephasing is to use a controlled 3' exonuclease. An example is Klenow polymerase which attempts to remove about 6 nucleotides at a time in a reaction without dNTPs. By repeating this process 3 to 5, 3 to 7, or 4 to 7 times, the desired number of nucleotides will be removed.

[0236] XI. Reversible blocking group

[0237] Deoxyribonucleotide analogs with reversible blocking groups are well known in the field of sequencing. Exemplary reversible blocking groups include amino-containing blocking groups (NH2-) (see Hutter et al., 2010, Nucleosides Nucleotides Nucleic Acids 29 (11)), allyl-containing blocking groups (e.g., CH2=CHCH2-); reversible blocking groups containing cyano groups (e.g., cyanovinyl or cyanoethyl); azido-containing blocking groups (N3-), such as azidomethyl (N3CH2-); alkoxy-containing blocking groups (e.g., CH3CH2O-). In some embodiments, the reversible blocking group comprises a polyethylene glycol (PEG) moiety having one or more ethylene glycol units. In some embodiments, the reversible blocking group is a substituted or unsubstituted alkyl, acyl (see U.S. Pat. No. 6,232,465), methoxymethyl; aminooxy (H2NO-), carbonyl (O=CH-), nitrobenzyl (C6H4(NO2)-CH2-), nitronapthyl, exemplary groups are described in U.S. Pat. No. 10,851,410. In some embodiments, nucleotides with non-removable (non-cleavable) 3' blocking groups can be used. In one method, after detection with an affinity reagent, the last incorporated base is removed and its position is filled with a similar nucleotide but with a cleavable blocking group (Koziolkiewicz et al., FEBS Lett. 434:77-82, 1998).

[0238] XII. Blocking group cleavage agents and conditions

[0239] As mentioned above, in some re-phasing methods, the incorporation of a reversible terminator is carried out at the first position of the selected dinucleotide pair, followed by unblocking cutting so that the second position is tested with a reversible terminator having an alternative 3' blocking group. This allows for continued selective unblocking of the first position until most of the readings have terminated at the selected dinucleotide pair. It is generally required that the unblocking of the second position should not be promoted by the unblocking agent at the first position, but the unblocking of the second position can allow the unblocking of the first position. Reversible terminator nucleotide analogs are well known in the art, and practitioners have many options for selecting combinations or pairs of blocking groups with non-overlapping conditions to be suitable for cutting in accordance with the present invention.

[0240] In addition to many chemical treatments with "non-overlapping" conditions, cleavage using enzymatic conditions, reducing conditions, oxidative conditions, or photocleavable conditions can interchangeably serve as the deblocking agent at position 1 or position 2.

[0241] Chemical treatment should not significantly degrade the template or primer extension chain. Various molecular moieties such as 3'-O-allyl groups for 3' blocking groups for reversible terminators have been described (Ju et al., Proc. Natl. Acad. Sci. USA 103: 19635-19640, 2006), 3'-O-azidomethyl-dNTPs (Guo et al., Proc. Natl Acad. Sci. USA 105, 9145-9150, 2008), aminoalkoxy (Hutter et al., N Nucleosides, Nucleotides and Nucleic Acids, 29: 879-895, 2010) and 3'-O-(2-cyanoethyl) groups (Knapp et al., Chem. Eur. J., 17, 2903-2915, 2011).

[0242] In one embodiment, a reducing agent such as phosphine THPP is used to unblock the first position (e.g., O-azidomethyl blocking group), and an oxidizing agent such as sodium nitrite is used to unblock the aminooxy group (Hutter et al., 2010 Labeled Nucleoside Triphosphates with Reversibly Terminating Aminoalkoxyl Groups. Nucleosides, Nucleotides & Nucleic Acids. 29, 879–895) at the second position. The blocking portion with '-O-allyl can be cleaved using a Pd catalyst generated by Na2PdCl4 and a phosphine ligand P(PhSO3Na)3 (TPPTS) that mediates deallylation. If the allyl group alone is resistant to phosphine cleavage, it can be used as a position 2 blocking group together with a phosphine-cleavable position 1 group such as an azidomethyl group (Ju et al., 2006, Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators. PNAS. 103, 19635–19640). The 3′-O-2-cyanoethyl (CE) group has been reported as a 3′ reversible terminator blocking group cleaved under alkaline conditions using tetrabutylammonium fluoride (TBAF) in THF and a small base (such as hydroxyl) (Keller et al., ChemInform Abstract: Synthesis of 3′-O-(2-Cyanoethyl)-2′-deoxythymidine-5′-phosphate as a Model Compound for Evaluation of Cyanoethyl Cleavage. Cheminform. 40 (2009), doi: 10.1002 / chin.200933204). In a similar manner, a cleavable 3' blocking group incorporating a disulfide bond that is cleavable under mild reducing conditions may be suitable as a position 1 blocking group, but a position 2 blocking group may require a stronger reducing agent such as a phosphine to cleave and / or benefit from specific salt and pH conditions for cleavage. Some methoxymethyl 3'-O reversible blocking groups may be cleaved with acid. 3'-O reversible blocking groups that may be cleaved by contact with a buffered aqueous solution of sodium nitrite (pH 5.5) include, but are not limited to, aminoalkoxy. Some 3'-O reversible blocking groups (e.g., nitrobenzyl) may be cleaved by UV light.Enzymatic cleavage mechanisms can also be used to remove phosphate blocking groups, such as with phosphatases (e.g., shrimp alkaline phosphatase, calf intestinal phosphatase, Antarctic phosphatase, and T4 polynucleotide kinase) and esterases (Canard et al., 1995, Catalytic editing properties of DNA polymerases. Proc Natl Acad Sci U S A. 92, 10859–10863). Photocleavable blocking groups compatible with chemical or enzymatic cleavage methods have also been described, such as 3′-O-nitrobenzyl (Metzker et al., 1994. Termination of DNA synthesis by novel 3′-modified-deoxyribonucleoside 5′-triphosphates. Nucleic Acids Res. 22, 4259–4267 (1994)).

[0243] XIII. Additional Embodiments

[0244] The above discussion describes in particular methods for re-phasing to a specified dinucleotide. The reader guided by the present disclosure will recognize that re-phasing can be designed to target a 3-base sequence (trinucleotide). On the one hand, for example, the present invention provides a method for re-phasing an extension primer in a clonal population of a nucleic acid duplex, wherein the clonal population of the nucleic acid duplex comprises an extension primer hybridized with a template sequence, wherein a plurality of extension primers in the clonal population have different 3' ends and are therefore out of phase, the method comprising extending the extension primer in the following manner: by using a polymerase and a nucleotide comprising a nucleotide triphosphate ester A, T, C and G or its analogues to incorporate a nucleotide complementary to the template sequence to reach a length of 1-3 (e.g., 3) nucleotides of the first target sequence, until substantially all extension primers reach the target sequence. Termination at a trinucleotide sequence comprises first terminating at a dinucleotide (as described in detail above), removing the second block, and continuing the process using a nucleotide or multiple nucleotides having the same or new nucleotides as the second block (for multiple different 3-mers selected for termination). For example, after terminating at the CA dinucleotide, we can continue to extend for 5 cycles, where A has the second blocking group and the other 3 nucleotides have the first blocking group cleaved after each 5 cycles. In this example, extension will terminate at these 3-mers: CA(noA) 0-5 A. If the second blocking group is used for T and C, the extension will be at CA (noA) 0-5 T and CA (noA) 0-5In one approach, 3mers are selected based on frequency in the sequence and tuned to stop extending at approximately every 10 bases, every 20 bases, every 25 bases, or every 30 bases.

[0245] In one method, the invention provides a method for re-phasing extension primers in a clonal population of nucleic acid duplexes, the clonal population of nucleic acid duplexes comprising extension primers hybridized to a template sequence, wherein multiple extension primers in the clonal population have different 3' ends and are therefore out of phase, the method comprising cutting the extension primer to the farthest target sequence of 1-4 bases in length within a predetermined window of 20, or 30, or 40 bases. The cutting can be used to re-phase the 3' or 5' end or both strands (cutting both strands at the target sequence). The 5'-cut can utilize a nicking enzyme with a known recognition sequence that can be attached to the 5' end with a linker that limits how far the enzyme can cut.

[0246] Example

[0247] Copy 1…BBBBCC'BBBBBBBBB-5'-Adapter-NE cut after CC

[0248] …BBBBGGBBBBBBBBBBBBBBB-3'

[0249] Copy 2…BBBBCC'BBBBBBB-5'-Adapter-NE cut after CC

[0250] ...BBBBGGBBBBBBBBBBBBBB-3'

[0251] NE indicates nickase. The linker can have a length that allows the nickase to cut at a site within 10 bases. Even when copy 1 has 9 bases from CC to the 5' end and copy 2 has 7 bases from CC to the 5' end, both copies will be rephased at CC.

[0252] Although embodiments in this disclosure are generally presented in the context of SBS sequencing, it is contemplated that the methods described herein may be used for a number of purposes, including but not limited to grouped ends produced by wobble restriction enzymes or incompletely synchronized primer extension or exonuclease degradation, or any other use in which members of a clonal population are out of phase.

[0253] The reader guided by the specification will recognize that minor variations may be made from the above description, all of which are envisioned by the inventors.The following hypothetical example is provided.

[0254]

[0255] In this hypothesis, a first scheme is shown, wherein the nucleotide is blocked by a first blocking group and a second blocking group. A second scheme shows a nucleotide blocked with a first blocking group, a second blocking group, a third blocking group, and a fourth blocking group, wherein the third and fourth blocking groups are equivalents or variants of the first blocking group and have the same properties relative to the second blocking group. It is understood that reference to "blocking group" for example includes functional equivalents (i.e., the first blocking group or a functional equivalent that shares properties with the first blocking group).

[0256] XIV. Polymerase

[0257] Any DNA polymerase for sequencing can be used in the methods disclosed herein, including, for example, a DNA polymerase from the genus Thermococcus, such as 9°N or a mutant thereof, including A485L, including double mutants Y409V and A485L. Exemplary DNA polymerases and methods that can be used include Chen, C., 2014, "DNA Polymerases Drive DNA Sequencing-By-Synthesis Technologies: Both Past and Present" Frontiers in Microbiology, Vol. 5, Article 305; Pinheiro, V. et al., 2012 "Polymerase Engineering: From PCR and Sequencing to Synthetic Biology" Protein Engineering Handbook: Volume 3: 279–302; International Patent Publications WO 2005 / 024010 and WO 2006 / 120433. Those DNA polymerases and methods described. In some cases, the polymerase is a DNA polymerase from Thermococcus, such as 9°N or a mutant thereof, including A485L, including the double mutants Y409V and A485L. Other examples include E. coli DNA polymerase I, Klenow fragment of DNA polymerase I, T7 or T5 phage DNA polymerase, HIV reverse transcriptase; Phi29 polymerase and Bst DNA polymerase.

[0258] XV. Reagent test kit

[0259] The present disclosure provides a kit or reagent combination for re-phasing. In one embodiment, the kit includes two reversible terminator nucleotide triphosphates, each of which has a different reversible blocking group, and the two reversible blocking groups are removable under different conditions.

[0260] For dinucleotide re-phasing, the kit may include a first nucleotide triphosphate (selected from A, T, C or G) and a second nucleotide triphosphate (independently selected from A, T, C or G), wherein the first nucleotide triphosphate is blocked by a first blocking group, and the second nucleotide triphosphate is blocked by a second reversible blocking group that is different from the first blocking group and removable under different conditions. Possible blocking groups include, but are not limited to, those listed above in Section XV. An example is a first blocking group of O-azidomethyl, and a second blocking group comprising an ONH2 group. The kit may also include a chemical reagent suitable for removing each blocking group at an appropriate time during re-phasing.

[0261] Alternatively, a test kit for re-phasing of dinucleotides may include a first nucleotide triphosphate (one to four selected from A, T, C or G) sealed with a first reversible blocking group, and an oligonucleotide, which is configured to hybridize with a template near a growth primer, typically at the 5' end of the oligonucleotide, optionally accompanied by a ligase suitable for connecting the oligonucleotide to the 3' end of a sequencing primer. As mentioned above, other positions of the oligonucleotide may be degenerate bases and / or universal bases. The oligonucleotide is usually sealed at the 3' end, but after encountering the dinucleotide selected for re-phasing, it can be removed from the growth primer. The test kit comprising such an oligonucleotide may also include a reagent for removing the first blocking group, and one or more reagents for removing the oligonucleotide, such as an enzyme mixture of uracil-DNA glycosylase (UDG) and apurinic / pyrimidinic endonuclease 1 (Apel) or similar abasic site endonuclease.

[0262] In some embodiments, the oligonucleotide has the following structure: 5′-Phos-U(N) Z -X, wherein "Phos" indicates that the oligonucleotide is 5' phosphorylated, "U" is uracil, Z is 6 to 20, preferably 6 to 12, preferably 9, "(N) Z " is a sequence of Z degenerate bases; and "X" is an irreversibly closed structure (including but not limited to dideoxynucleotides or inverted bases).

[0263] Any of these kits may also contain one or more reagents for repositioning the growing chain by backtracking: for example, uracil triphosphate (with or without UDG and Apel), nucleotides with RNA or thiolated bases, and / or endonucleases or exonucleases.

[0264] More comprehensive kits include any of these reagent combinations for re-phasing and / or repositioning extended sequencing primers in conjunction with other reagents for synthesis sequencing: for example, reversible terminators with directly attached fluorophores or labeled antibodies, DNA polymerases, and reagents for preparing concatemers, DNB arrays, bridged PCR strands, and other clonal populations of DNA fragments to be sequenced.

[0265] The reagents in these kits are typically supplied individually, or in working mixtures in standard containers or in modules specifically designed to pump the reagents into a sequencing device or flow cell. According to the present disclosure, the reagents are optionally distributed with or in conjunction with instructions for using the reagents in sequencing and re-phasing.

[0266] XVI. Computer simulation (IN SILICO) Rephasing

[0267] The technology proposed in this disclosure was modeled by computer simulation to demonstrate its effectiveness in reducing the inconsistency of growing chains and increasing the effective read length. In the above described re-phasing option, the process used in this simulation includes the following steps:

[0268] (1) Backtracking 30 bases, for example, can be accomplished by going back 30 cycles in sequencing to introduce uracil into the growing chain and cutting with a UDG / Ape1 enzyme mixture corresponding to the A base in the sequence;

[0269] (2) Rephasing events using the dinucleotide frequency rephasing process described in "Method 2" above. Simulations were performed using alternative dinucleotides CA or CG. Each rephasing cycle involves extending the non-closed chain to the first base in the pattern (C), and then inserting a blocking agent if the next base is the second base in the rephasing dinucleotide (A or G). This can be shown as follows:

[0270] AACTACAGCTGC—Initial starting position

[0271] New P: AACTACAGCTGC—the read position is shifted back about 10 bases

[0272] Step 1: AACTACAGCTGC—extend to first C, next base is not A

[0273] Step 2: AACTACAGCTGC—extend to next C, add blocking agent to A

[0274] Step 3: AACTACAGCTGC—stays in A due to blocking agent

[0275] A. Predicting reaction conditions

[0276] In practice, the underlying technology used for the in silico experiments can be implemented as follows: DNA nanoballs (DNBs), concatemers of nucleic acid templates to be sequenced, are arrayed on a solid surface and analyzed by sequencing by synthesis, determining each base sequentially guided by the complementary strand.

[0277] DNB is generated by amplification cascades to produce single-stranded multiple repeats of reverse complementary single-stranded circles as described previously (R. Drmanac et al., Science. 327, 78-81, 2000). The DNB array is sequenced by gradually adding 3'-blocked reversible termination nucleotides with DNA polymerase, followed by detection with fluorescently labeled antibodies ("CoolMPS", U.S. Patent No. 10,851,410). The 3' reversible termination group is O-azidomethyl (AzM), which is unblocked with 10mM tris(hydroxypropyl)phosphine (THPP) at 55°C for 2 minutes. The 3' blocking group is cleaved into the 3' hydroxyl group to allow continuous incorporation for sequential base determination.

[0278] B. Error Simulation

[0279] To perform the in silico simulations, sequencing was assumed to proceed over multiple sequencing-by-synthesis cycles, and computer-generated errors were imputed to the data in each sequencing cycle at approximately the same frequency known to occur in live flow cell sequencing. Assuming the backoff and rephasing events were completed, the effect of rephasing was determined.

[0280] The simulation parameters are as follows: DNB count: 977029, copy number (number of copies per DNB fragment): 180 (CV: 20%). Two target sequences are used: one, a portion of the human genomic DNA reference sequence; the second, a computer-generated random sequence with approximately the same total composition: A and T, 27%; C and G, 23%. The sequencing simulation samples regions from the reference genome and models the phasing and labeling randomness of the array of incorporation sites corresponding to independent copies on each DNB. The label sites are clustered into corresponding channels based on the sequence context of the site position. This produces an array containing the number of label sites present in each channel in each cycle.

[0281] The sequencing errors introduced were 0.1% hysteresis, 0.05% running and 0.15% termination. Hysteresis and running cause the copy being sequenced to diverge, while termination stops further chemical processing of that copy, effectively resulting in a reduction in the intensity from the acceptor DNB.

[0282] Five or seven rephasing cycles were simulated for each rephasing event. Two rephasing events were performed after 300 or 600 bases sequenced, or three rephasing events were performed after 225, 450, and 675 bases sequenced.

[0283] Simulate output data based on fluorescent labels detected for each primer in each DNA nanoball arranged in a grid pattern in each cycle. The grid can be adjusted for different distances between DNBs and different pixel resolutions. The normal distribution of pixel values ​​is added to the image to simulate the impact of background. The results shown are based on typical distances, distributions and backgrounds of DNA sequence devices for nanoball sequencing.

[0284] C. Fallback Steps Using Uracil

[0285] Because forward run incorporation during rephasing creates gaps in the sequencing data under these conditions, a backoff process was first used to remove already sequenced DNA fragments to ensure that sequence coverage was not lost. The computer model assumed a backoff of 30 residues before each rephasing event.

[0286] In practical applications, this can be done as follows. 30 cycles before synthesis sequencing stops, the standard synthesis sequencing reagent mixture is switched to a mixture containing 3 μM each of dUTP-AzM, dATP-AzM, dCTP-AzM, dGTP-AzM and DNA polymerase. Due to this switch, dUTP-AzM replaces dTTP-AzM, and sequencing continues for another 30 cycles using the alternative spike-in mixture. Antibodies commonly used to recognize dTTP-AzM nucleotides can also be used in cycles containing dUTP, provided that it has sufficient cross-reactivity and specificity to recognize dUTP-AzM nucleotides.

[0287] After the last 30 cycles of sequencing, the extended primers were treated with a mixture of uracil-DNA glycosylase (UDG) enzyme (2 U / μL) and apurinic / apyrimidinic endonuclease 1 (Ape1) endonuclease (1 U / μL) at 37°C for 10 minutes to cleave the uracil base and the subsequently generated abasic site. The effect was to backtrack the extended and sequenced DNA strands to the first uracil incorporated in the 30 incorporation cycles using dUTP-AzM. After buffer exchange to remove the UDG / Ape1 mixture, the flow cell was washed multiple times in low salt buffer at 55°C to remove short cleavage sequences.

[0288] D. Dinucleotide Rephasing Step

[0289] After backing off, the computer model assumes that the primer will be extended again until the selected dinucleotide is recognized and blocked.

[0290] In actual practice, this can be done as shown in Table 1.

[0291]

[0292] An incorporation mixture consisting of 1 reversible terminator dCTP-AzM and 3 unblocked natural nucleotides dATP, dGTP and dTTP is generated. Typically, the concentration of the nucleotide is 3M, accompanied by a DNA polymerase: for example, a DNA polymerase variant capable of incorporating an azidomethyl 3' blocking group and natural nucleotides (U.S. Patent No. 10,851,410). The incorporation time (step 1A) is typically 2 minutes and is performed at a temperature of 55°C. Natural nucleotides can be freely incorporated in a sequential manner dictated by the template sequence, but when dCTP-AzM is incorporated, extension is terminated due to the 3' blocking group. After the first incorporation step, cleavage of the incorporated dCTP-AzM group is performed by incubating with THPP at a concentration of 10 mM for 2 minutes. Likewise, the reaction temperature is maintained at 55°C.

[0293] Cleavage results in the conversion of the 3' blocking group to a 3' hydroxyl group, which allows further incorporation of terminal cytosine. The second incorporation reaction (step 1B) includes 3'-aminooxy-modified dATP (dATP-ONH2) as the blocking nucleotide, dCTP-AzM, dGTP-AzM, dTTP-AzM nucleotides and 9 0 N variant DNA polymerase. A second cleavage was performed with 10 mM THPP to specifically unblock the incorporated C, G, and T nucleotides for further extension. Those terminal C bases that incorporate dATP-ONH2 as the next base resist cleavage by THPP and are therefore blocked for further extension.

[0294] To complete the re-phasing event, steps 1A and 1b were repeated four more times. Step 6 was to block all incorporated and accumulated dATP-ONH2 nucleotides with 700 mM sodium nitrite at 25°C for 2 minutes. Since most DNB copies were now in phase, sequencing calls could begin again.

[0295] E. Simulation Results

[0296] The parameters and error variables mentioned in subsection "B" above were implemented in the computer model and the results are as follows.

[0297] Figure 2The percentage of DNB templates in which 100% of the copies or subunits were rephased back to the reference sequence under different rephasing conditions is shown. Each triplet represents the extent of rephasing after the first, second, or third rephasing event. Using CA as a rephasing dinucleotide is slightly more efficient than using CG; seven (7) rephasing cycles are slightly more efficient than 5 rephasing cycles; three rephasing events are slightly more efficient than two rephasing events. For human sequences, the difference between CA dinucleotides and CG dinucleotides is more obvious because CG occurs less frequently in the human genome than CA. For random simulated sequences (CA: approximately 6.21% vs. CG: approximately 5.29%), CA and CG do not occur at exactly equal frequencies, although they are significantly closer in frequency compared to the human reference (CA: approximately 7.27% vs. CG: approximately 0.99%).

[0298] Remarkably, all conditions resulted in more than 85% of DNBs being re-phased. Without any re-phasing, the number of in-phase DNBs was close to zero. Of the approximately one million DNBs on the array, only two had more than 95% in-phase spots at different re-phasing cycles. At sequencing cycle 225, one DNB had one site at the minus 1 position and 35 in-phase spots. At sequencing cycle 675, one DNB had only one active site in phase.

[0299] Figure 3 Displays the synchronization percentage of DNB. This data reveals Figure 2 The reason for the increase in the percentage of DNBs that are 100% in phase between rephasing events is shown. One explanation for this trend is that the stochastic nature of termination leads to the elimination of out-of-phase positions for DNBs that were not fully in phase during the previous rephasing event. This would result in DNBs that were not 100% in phase previously being transformed by only one pair of sites to have a higher probability of being fully in phase during the next round of rephasing. Figure 3 The data in show how the percentage of DNBs with only one site out of phase is inversely proportional to the percentage of DNBs that are completely in phase. While the percentage of DNBs that are completely synchronized increases between re-phasing events, the percentage of DNBs with only one site out of phase decreases. When the two percentages are added together, the percentage of DNBs with <= 1 site out of phase remains consistent between re-phasing events.

[0300] The statistics for the different rephasing conditions are compared in Table 2. The statistics in bold and / or underlined in the table are slightly superior. All conditions were valid.

[0301] Figure 4A The kinetics of phase inconsistency of the growing chains between the different parameters tested on the human reference sequence were compared. Figure 4Bis a similar comparison of a randomly generated reference sequence. Under simulated conditions, without the need for re-phasing, inconsistencies accumulated rapidly after the 300th sequencing cycle and were greater than 5% at 500 cycles. Two re-phasing events maintained inconsistencies below 2% for more than 750 cycles. Three re-phasing events maintained inconsistencies below 2% for more than 900 cycles. The extent of inconsistency is also shown in Table 3.

[0302] Figure 5 Cumulative cycle shift after the final re-phasing event is shown. This is a survival curve where the x-axis corresponds to the expected cumulative cycle shift after the last re-phasing event. Negative values ​​will correspond to the generation of overlapping sequence regions during re-phasing, while positive values ​​will correspond to sequencing through the allocated number of cycles. In the best case, the CG dimer pattern will result in 40% of DNBs being sequenced through the endpoint, while the CA dimer pattern will only have about 8% of DNBs. These curves illustrate that it may be necessary to have a small buffer after the sequencer cycles are completed to prevent a large number of DNBs from being sorted as adapters (e.g., 950 bases for 900 sequencing cycles).

[0303] It is clear that the dimer pattern used for rephasing plays a key role in the percentage of DNBs that will eventually order beyond the number of assigned bases. This can affect both the number of DNBs that order into the adaptor region and the length of overlapping sequences generated during the rephasing process.

[0304] ***

[0305] The invention has been described with reference to specific embodiments and illustrations in this disclosure. Unless expressly stated or otherwise required, the features of these embodiments and illustrations do not limit the implementation of the claimed invention. As a matter of routine development and optimization and within the scope of ordinary technicians in the field, changes can be made and equivalents can be substituted to adapt to specific contexts or intended uses, thereby achieving the benefits of the present invention without departing from the scope of the claimed protection and its equivalents.

[0306] Each publication and patent document mentioned in this disclosure is herein incorporated by reference in its entirety for all purposes in the United States to the same extent as if each such publication and patent document was specifically and individually indicated to be incorporated by reference.

[0307]

[0308] Table 3: Comparison of inconsistencies in rephasing simulations

[0309] refer to step# Doublet event average C0-300 C300-600 C600-900 people 7 CA 3 0.38 0.003 0.114 1.033 people 7 CG 3 0.37 0.005 0.128 0.989 people 5 CA 3 0.42 0.003 0.123 1.125 people 5 CG 3 0.40 0.005 0.136 1.058 people 5 CA 2 0.77 0.012 0.353 1.936 people 5 CG 2 0.74 0.013 0.386 1.812 people * * 0 8.18 0.013 3.229 21.322 people 7 CA 3 0.33 0.004 0.102 0.885 people 7 CG 3 0.36 0.004 0.116 0.949 people 5 CA 3 0.38 0.004 0.119 1.011 people 5 CG 3 0.39 0.004 0.130 1.050 people 5 CA 2 0.70 0.012 0.344 7.757 people 5 CG 2 0.73 0.012 0.367 1.809 people * * 0 8.28 0.012 3.102 21.740

[0310] (*) = no rephasing.

Claims

1. A method for re-phasing extension primers in a clonal population of nucleic acid duplexes comprising extension primers hybridized to a template sequence, wherein a plurality of said extension primers in said clonal population have different 3' ends and are therefore out of phase, said method comprising: (1) extending the extension primer by incorporating one or more nucleotides complementary to the template sequence using a polymerase and a nucleotide comprising the nucleotide triphosphates A, T, C and G, wherein one of the nucleotides is a blocking nucleotide blocked with a first blocking group and the other three nucleotides are not blocked until substantially all of the extension primer is blocked; then (2) unblocking the blocked nucleotide incorporated into the extension primer to form an unblocked nucleotide, Wherein the re-phasing comprises dinucleotide frequency re-phasing DFR, wherein each extension primer is extended until a selected dinucleotide XY is reached.

2. The method according to claim 1, wherein the first nucleotide X is a blocked nucleotide blocked by a first blocking group, and the second nucleotide Y is a blocked nucleotide blocked by a second blocking group.

3. The method according to claim 1, wherein any one of the first blocking group and the second blocking group is an O-azidomethyl group, and the other of the first blocking group and the second blocking group is an O-NH2 group. 4 . The method of claim 1 , wherein 5 to 50 bases are removed from the 3′ end of each primer prior to the re-phasing, thereby realigning the 3′ end of the extension primer to an upstream position.

5. The method of claim 4, wherein the readjusting comprises: (i) during sequencing by synthesis performed prior to said re-phasing, which includes in at least some cycles of said sequencing a uracil triphosphate which can be incorporated into said extension primer in place of a thymine triphosphate; and then (ii) cleavage of the extension primer at the incorporated uracil base.

6. The method according to claim 5, wherein the cleavage in step (ii) is performed using an enzyme mixture of uracil DNA glycosylase UDG and apurinic / apyrimidinic endonuclease Apel.

7. The method of claim 4, wherein the readjusting comprises: (i) during sequencing by synthesis performed prior to said re-phasing, including in at least some cycles of said sequencing nucleotide triphosphates containing RNA or 5' alpha-phosphothioate modified nucleotides; and then (ii) cleaving the extension primer at an incorporated RNA base or an incorporated 5' α-phosphothioate modified nucleotide.

8. The method of claim 4, wherein the readjustment comprises treating the extension primer with a 3' exonuclease under controlled conditions, or treating the extension primer with a subsequence-dependent nickase, thereby removing the 5 to 50 bases from the 3' end of the extension primer.

9. The method of any one of claims 1 to 8, further comprising restarting a sequencing cycle after said re-phasing, whereby said extension primers in said clonal population are extended by bases, each base recognizing a complementary nucleotide in said template sequence.

10. A method of re-phasing extension primers in a clonal population of nucleic acid duplexes to a selected dinucleotide XY, wherein each of the duplexes comprises an extension primer that anneals to a template sequence, wherein the plurality of extension primers in the clonal population have different 3' ends and are therefore out of phase, The method comprises: (a) Perform multiple cycles of the following: (i) extending the extension primer using a first mixture comprising a polymerase and four nucleotide triphosphates selected from A, T, C and G, wherein one of the nucleotide triphosphates in the first mixture corresponds to the first nucleotide X of the selected dinucleotide and is a blocking nucleotide blocked by a first blocking group, and wherein the other three nucleotide triphosphates in the first mixture are not blocked, continuing the extension until substantially all of the extension primer is blocked by the first blocking group; and then (ii) unblocking the first blocking group; and (iii) treating the extended primer from (ii) with a second mixture containing a polymerase and a second nucleotide Y corresponding to the selected dinucleotide and being a single blocking nucleotide selected from A, T, C or G blocked by a second blocking group, wherein the second mixture optionally includes three nucleotide triphosphates that do not correspond to the second nucleotide Y blocked by the first blocking group, (b) repeating step (a) until substantially all of the extension primers are blocked by the second blocking group; and (c) unblocking the second blocking group; The extension primers in the clonal population are thereby rephased.

11. The method of claim 10, wherein the only nucleotide triphosphates in the second mixture are blocked nucleotides blocked by the second blocking group.

12. The method according to claim 10, wherein the second mixture contains the blocked nucleotide blocked by the second group, and the three nucleotide triphosphates that do not correspond to the second nucleotide Y blocked by the first blocking group.

13. The method according to claim 10, wherein any one of the first blocking group and the second blocking group is an O-azidomethyl group, and the other of the first blocking group and the second blocking group is an O-NH2 group.

14. A method of re-phasing extension primers in a clonal population of nucleic acid duplexes to a selected dinucleotide XY, wherein each of the duplexes comprises an extension primer that anneals to a template sequence, wherein a plurality of said extension primers in said clonal population have different 3' ends and are therefore out of phase, The method comprises: (a) Perform multiple cycles of the following: (i) extending the extension primer using a first mixture comprising a polymerase and four nucleotide triphosphates selected from A, T, C and G, wherein one of the nucleotide triphosphates in the first mixture corresponds to the first nucleotide X of the selected dinucleotide and is a blocking nucleotide blocked by a first blocking group, and wherein the other three nucleotide triphosphates in the first mixture are not blocked, continuing the extension until substantially all of the extension primers are blocked by the first blocking group; and then (ii) unblocking the first blocking group; and (iii) treating the extension primer with a second mixture comprising a ligase and a 5' phosphorylated oligonucleotide blocked at the 3' end, wherein the base in the oligonucleotide corresponds to the second nucleotide Y of the selected dinucleotide; (b) repeating step (a) until substantially all of said extension primers are blocked by said oligonucleotide; and (c) unblocking the oligonucleotide; The extension primers in the clonal population are thereby rephased.

15. The method of claim 14, wherein the 5' phosphorylated oligonucleotide has the formula U(N) Z -X, wherein U is uracil, X is a closed structure, Z is 6-20, and (N) Z is a degenerate oligonucleotide sequence, and wherein said unblocking in step (c) comprises removing said oligonucleotide from said extension primer.

16. The method according to claim 15, wherein: The blocking structure incorporates an inverted dT at the 3' end of the oligonucleotide, thereby creating a 3'-3' bond that inhibits degradation by 3' exonucleases and extension by DNA polymerases.

17. The method of claim 15, wherein the 5' phosphorylated oligonucleotide is removed by treatment with an enzyme cocktail of uracil DNA glycosylase UDG and apurinic / apyrimidinic endonuclease Apel to cleave and remove the uracil base.

18. The method of claim 15, wherein five to fifteen cycles are performed in step (a).

19. The method of any one of claims 14 to 18, further comprising restarting a sequencing cycle after said re-phasing, whereby said extension primers in said clonal population are extended by bases, each base recognizing a complementary nucleotide in said template sequence.

20. A method for obtaining long sequencing reads from a clonal population of nucleic acid duplexes, each nucleic acid duplex comprising an extension primer annealing to a template sequence, the method comprising: performing a plurality of cycles of sequencing, wherein the extension primer in each duplex is extended by one nucleotide, thereby identifying a complementary nucleotide in the template sequence; After a plurality of such sequencing cycles, re-phasing the extension primer according to the method of any one of claims 1 to 18; Then The sequencing cycles are resumed to identify additional nucleotides in the template sequence.

21. The method of claim 20, wherein the re-phasing is performed 2 to 4 times within the first 800 sequencing cycles.

22. The method according to claim 20, wherein: The rephasing expanded the number of clonal populations with a discordance percentage less than 2% by at least 1.5-fold.

23. The method of claim 20, wherein the rephasing expands the number of clonal populations having a discordance percentage of less than 2% for at least 200 cycles.

24. A sequencing method, comprising: providing an array comprising a plurality of clonal populations, each clonal population comprising a nucleic acid duplex wherein a sequencing primer anneals to a template sequence; performing multiple rounds of sequencing by synthesis to extend said sequencing primers until at least some of said primers have 3' ends that are different from, and therefore out of phase with, other primers in the same said clonal population; Rephasing the primers according to the method of any one of claims 1 to 18; Then The sequencing-by-synthesis is resumed after the re-phasing.

25. The method of claim 23, wherein each clonal population is a copy of a template sequence in a cluster of DNA strands or an emulsion droplet.

26. The method of claim 24, wherein the re-phasing is performed 2 to 4 times during the sequencing, thereby obtaining a read length of at least 800 bases.

27. A method for re-phasing multiple copies of a nucleic acid to be sequenced, wherein each copy comprises a single-stranded nucleic acid template hybridized to an extendable oligonucleotide sequencing probe, the method comprising: (1) extending the probe in a complementary manner to the template using a polymerase and a mixture of nucleotide triphosphates, wherein one of the nucleotide triphosphates is a 3' reversibly blocked nucleotide triphosphate, until substantially all of the copies are blocked; and then (2) unblocking the reversibly blocked nucleotide incorporated into the extension probe.

28. The method of re-phasing according to claim 27, which is a dinucleotide frequency re-phasing (DFR) method, comprising: (a) Perform multiple cycles of the following: extending the probe in a complementary manner to the template using a polymerase and a mixture of nucleotide triphosphates, wherein one of the nucleotide triphosphates is a nucleotide triphosphate blocked by a first blocking group, until substantially all copies are blocked; and then unblocking the first blocking group; and The probe is extended in a complementary manner to the template using a polymerase and a nucleotide triphosphate blocked with a second blocking group under conditions that add only a single nucleotide to the primer; then (b) unblocking the first blocking group and the second blocking group.

29. The method of claim 28, wherein 5 to 15 cycles are performed.

30. The method of claim 28, wherein the first blocking group is an O-azidomethyl group or an ONH2 group, and the second blocking group is an ONH2 group or an O-azidomethyl group, respectively.

31. The method of re-phasing according to claim 27, which is a method of dinucleotide frequency re-phasing (DFR), comprising: (a) Perform multiple cycles of the following: Extending the probe in a complementary manner to the template using a polymerase and a mixture of nucleotide triphosphates, wherein one of the nucleotide triphosphates is a nucleotide triphosphate blocked by a first blocking group, until substantially all copies are blocked; and then unblocking the first blocking group; and The probe is extended in a complementary manner to the template by attaching a 5' phosphorylated oligonucleotide to the sequencing probe, the 5' phosphorylated oligonucleotide being blocked at the 3' end by a second blocking group; and then (b) unblocking the 5' phosphorylated oligonucleotide.

32. The method of claim 31, wherein the 5' phosphorylated oligonucleotide has the formula UN 1-15 X, where U is uracil, N 1-15 is a sequence of 1 to 15 degenerate bases; X is an irreversible closed structure.

33. The method of claim 32, wherein the 5' phosphorylated oligonucleotide is unblocked by treatment with UDG / Ape or a similar abasic site endonuclease mixture to cleave and remove the uracil base, leaving an unblocked T stop base.

34. The method for re-phasing according to any one of claims 27 to 33, further comprising realigning sites for further sequencing before step (1).

35. The method of claim 34, wherein said re-adjusting said site comprises: (i) extending the probe in a complementary manner to the template using a polymerase and a mixture of nucleotide triphosphates, wherein one of the nucleotide triphosphates is uracil; and then (ii) Cleavage of the probe at the incorporated uracil base.

36. The method of claim 34, wherein: The re-adjusting the site comprises: (i) extending the probe in a complementary manner to the template using a polymerase and a mixture of nucleotide triphosphates, one of which is an RNA base; and then (ii) Cleavage of the probe at the incorporated RNA base.

37. The method of claim 34, wherein said re-adjusting said site comprises treating said probe with a controlled 3' exonuclease.

38. The method of claim 34, wherein said re-adjusting said site comprises treating said probe with a nicking enzyme in a sequence-dependent manner.

39. The method of any one of claims 27 to 33, further comprising restarting the sequencing cycle, thereby extending the sequencing probe in each copy to identify complementary nucleotides in the template.

40. A method for obtaining long sequencing reads from multiple copies of a target nucleic acid, wherein each copy comprises a single-stranded nucleic acid template hybridized to an extendable oligonucleotide sequencing probe; the method comprising: performing multiple rounds of sequencing, wherein the sequencing probe in each copy is extended by one nucleotide to thereby identify a complementary nucleotide in the template; After a plurality of such cycles, the nucleic acid copies are rephased according to the method of any one of claims 27 to 38; and then The sequencing cycles are resumed to identify additional nucleotides in the template.

Images