阅读说明：本技术 在体外克隆中分选核酸和多重制备物的方法 (Method for sorting nucleic acids and multiplex preparations in vitro cloning ) 是由 J·雅各布森 M·J·戈德伯格 L-Y·A·昆 D·辛德勒 M·E·赫德森 于 2013-04-24 设计创作，主要内容包括：方法和组合物涉及使用高通量测序进行的高保真核酸的分选和克隆。具体地,可从包含具有正确和错误序列的多种核酸的集合中分选具有需要的预定序列的核酸分子。(Methods and compositions relate to high fidelity sorting and cloning of nucleic acids using high throughput sequencing. In particular, nucleic acid molecules having a desired predetermined sequence can be sorted from a collection comprising a plurality of nucleic acids having correct and incorrect sequences.)

1. A method of sorting nucleic acid molecules having a predetermined sequence, the method comprising:

(a) providing a collection of nucleic acid molecules comprising at least two populations of nucleic acid molecules, each population of nucleic acid molecules having

(i) A unique target nucleic acid sequence that is the same for each population, the target nucleic acid sequence having two ends;

(ii) a pair of non-target oligonucleotide tag sequences located at each end of each target nucleic acid molecule, whereby each oligonucleotide tag sequence in the collection of nucleic acid molecules comprises a unique portion for each target nucleic acid sequence; wherein the collection is prepared by tagging target nucleic acid molecules with a library of non-target oligonucleotide tag sequences, the number of non-target oligonucleotide tag sequences being greater than the number of target nucleic acid molecules;

(b) sequencing the target nucleic acid molecule starting from both ends resulting in paired end reads; and is

(c) Nucleic acid molecules having a predetermined sequence are sorted according to their corresponding paired end reads.

2. The method of claim 1, further comprising amplifying the nucleic acid molecule having a predetermined sequence.

3. The method of claim 2, further comprising amplifying the nucleic acid molecule having the predetermined sequence with a primer that is complementary to at least a portion of the oligonucleotide tag sequence.

4. The method of claim 1, wherein the target nucleic acid molecules comprise a first population of target nucleic acid molecules having the predetermined sequence and a second population of target nucleic acid molecules having a sequence different from the predetermined sequence.

5. The method of claim 4, further comprising assembling a plurality of target nucleic acid molecules on a solid support and pooling the target nucleic acid molecules.

6. The method of claim 5, further comprising diluting the target nucleic acid molecule.

7. The method of claim 1, further comprising diluting the collection of nucleic acid molecules.

8. The method of claim 1, wherein the non-target oligonucleotide tag sequences are attached to both ends of each target nucleic acid molecule.

9. The method of claim 8, wherein the non-target oligonucleotide tag sequence is added to the target nucleic acid molecule by ligating the target nucleic acid molecule to one or more nucleic acid molecules comprising the non-target oligonucleotide tag sequence.

10. The method of claim 9, wherein the one or more nucleic acid molecules comprising the non-target oligonucleotide tag sequence is a vector.

11. The method of claim 1, wherein in the step of providing tagging, the non-target oligonucleotide tag sequences are attached to both ends of each of the target nucleic acid molecules by polymerase chain reaction.

12. The method of claim 1, wherein each non-target oligonucleotide tag sequence comprises a degenerate nucleotide sequence or a partially degenerate sequence.

13. The method according to claim 12, wherein the degenerate nucleotide sequence is ccwswdhshdbvhdnnmm and/or ccswswhdhvhdhnnnnmm, wherein W represents a or T, S represents G or C, M represents a or C, B represents C, G or T, D represents A, G or T, H represents A, C or T, V represents A, C or G, and N represents any base A, C, G or T.

14. The method of claim 2, wherein in the amplifying step, the primers are complementary to a pair of non-target oligonucleotide tag sequences.

Technical Field

The methods and compositions of the present invention relate to nucleic acid assembly, and in particular to methods of sorting and cloning nucleic acids having a predetermined sequence.

Background

Recombinant and synthetic nucleic acids have many applications in research, industry, agriculture, and medicine. Recombinant and synthetic nucleic acids can be used to express and obtain a wide variety of polypeptides, including enzymes, antibodies, growth factors, receptors, and other polypeptides useful for a variety of medical, industrial, or agricultural purposes. Recombinant and synthetic nucleic acids can also be used to produce genetically modified organisms, including modified bacteria, yeast, mammalian, plant, and other organisms. Genetically modified organisms can be used in research (e.g., animal models of disease, tools to understand biological processes, etc.), industry (e.g., as host organisms for protein expression, bioreactors for producing industrial products, tools for environmental remediation, isolating or modifying natural compounds with industrial applications, etc.), agriculture (e.g., modified crops with increased yield or increased resistance to disease or environmental stress), and for other applications. Recombinant and synthetic nucleic acids can also be used as therapeutic compositions (e.g., for modifying gene expression, for gene therapy, etc.) or as diagnostic tools (e.g., probes for disorders, etc.).

Various techniques have been developed for modifying existing nucleic acids (e.g., naturally occurring nucleic acids) to produce recombinant nucleic acids. For example, nucleic acid amplification, mutagenesis, nuclease digestion, ligation, cloning, and other techniques can be used to produce many different recombinant nucleic acids. Chemically synthesized polynucleotides are often used as primers or adapters for nucleic acid amplification, mutagenesis, and cloning.

Techniques for de novo nucleic acid assembly have also been developed, in which nucleic acids are prepared (e.g., chemically synthesized) and assembled to produce longer target nucleic acids of interest. For example, different multiplex assembly techniques have been developed for assembling oligonucleotides into larger synthetic nucleic acids that can be used in research, industry, agriculture, and/or medicine. However, one limitation of currently available assembly techniques is the relatively high error rate. Therefore, a high fidelity, low cost assembly method is needed.

Disclosure of Invention

Aspects of the invention relate to methods of sorting and cloning nucleic acid molecules having a desired or predetermined sequence. In some embodiments, the method comprises providing one or more pools of nucleic acid molecules comprising at least two populations of nucleic acid molecules of interest, each population of nucleic acid molecules having a unique nucleic acid sequence of interest, tagging the 5 'ends and 3' ends of the nucleic acid molecules with a non-target oligonucleotide tag sequence, wherein the oligonucleotide tag sequence comprises a unique nucleotide tag and a primer region, diluting the tagged nucleic acid molecules, performing a sequencing reaction on the tagged nucleic acid molecules starting from both ends to obtain paired end reads, and sorting the nucleic acid molecules having the desired sequence according to the identity of their corresponding unique oligonucleotide tag pairs. In other embodiments, the method comprises providing one or more collections of nucleic acid molecules comprising at least two populations of nucleic acid molecules, each population of nucleic acid molecules having a unique internal nucleic acid sequence and, at its 5 'and 3' ends, an oligonucleotide tag sequence comprising a unique nucleotide tag and a primer region, performing a sequencing reaction on the tagged nucleic acid molecules starting from both ends to obtain paired end reads, and sorting the nucleic acid molecules having the desired sequence according to the identity of their respective unique oligonucleotide tag pairs. In some embodiments, each population of nucleic acid molecules has a different desired nucleic acid sequence.

In some embodiments, unique nucleotide tags may be attached to the 5 'end and the 3' end of a nucleic acid molecule. In other embodiments, a unique oligonucleotide tag can be attached to each end of a nucleic acid molecule by PCR. In some embodiments, a unique nucleotide tag can include a fully degenerate sequence, a partially degenerate sequence, or a non-degenerate sequence. In some embodiments, the unique oligonucleotide tag may comprise an encoded barcode. For example, the unique nucleotide tag may include the following sequence: CCWSWDHSHDBVHDNNMM or CCSWHDSDHVBDHNNNNMM.

In some embodiments, the method further comprises amplifying the nucleic acid molecule having the desired sequence. In some embodiments, the method comprises amplifying the construct having the desired sequence using primers complementary to the primer region and the tag nucleotide sequence. In some embodiments, the method comprises amplifying the construct having the desired sequence using a primer complementary to the oligonucleotide tag sequence. In other embodiments, a primer complementary to a target nucleic acid sequence can be used to amplify a construct having a desired sequence.

In some embodiments, the method further comprises pooling a plurality of nucleic acid molecules to form a collection of nucleic acid molecules, wherein each of the plurality of nucleic acid molecules comprises a population of nucleic acid sequences having a desired sequence (i.e., an error-free nucleic acid sequence) and a population of nucleic acid sequences that is different from the desired sequence (i.e., an error-containing nucleic acid sequence). In some embodiments, the nucleic acid molecule can be assembled de novo. In some embodiments, the plurality of nucleic acid molecules may be diluted prior to or after the pooling step to form a normalized collection of nucleic acid molecules.

In some embodiments, the oligonucleotide tag may be attached to the nucleic acid molecule prior to diluting the nucleic acid molecule from the collection. In some embodiments, the method may further comprise amplifying the tagged nucleic acid molecule after the dilution step. In other embodiments, the oligonucleotide tag may be attached to the nucleic acid molecule after dilution of the nucleic acid molecule from the collection.

In some embodiments, each nucleic acid molecule comprises a 5 'end common adaptor sequence and a 3' end common adaptor sequence and the oligonucleotide tag sequence further comprises a common adaptor sequence. In some embodiments, each nucleic acid molecule is designed to have a 5 'end common adaptor sequence and a 3' end common adaptor sequence. In other embodiments, a 5 'end common adaptor sequence and a 3' end common adaptor sequence are added to each nucleic acid molecule by a ligation reaction.

Some aspects of the invention relate to methods of designing a plurality of oligonucleotides to assemble into a nucleic acid sequence of interest having a predetermined sequence. In some embodiments, the method comprises computationally partitioning each nucleic acid sequence of interest into partially overlapping construction oligonucleotide sequences; selecting a first set of construction oligonucleotide sequences such that every two adjacent construction oligonucleotide sequences overlap each other by N bases, wherein each N-base sequence is at least 4 bases in length; comparing the N-base sequences with each other to satisfy one or more of the following constraints: the N-base sequences differ from each other by at least 2 bases, or the N-base sequences differ from each other by at least 1 base in the last 3 bases of the 5 'end or the 3' end; identifying from the first set of construction oligonucleotide sequences a second set of construction oligonucleotide sequences that satisfies the constraint; determining the number of oligonucleotides in the second oligonucleotide set; ranking the oligonucleotides from the second oligonucleotide set that meet or exceed the constraints and based on the number of oligonucleotides; and this ranking is used to design a satisfactory set of partially overlapping construction oligonucleotides. In the ranking step, groups with a smaller number of oligonucleotides may be selected, and/or groups with a higher number of base differences in the N-base sequence may be selected. In some embodiments, a non-target flanking sequence may be added to the end of at least a portion of the construction oligonucleotide by computer. The non-target flanking sequences may comprise primer binding sites. The method also includes synthesizing a satisfactory set of partially overlapping construction oligonucleotides, e.g., on a solid support, and assembling the construction oligonucleotides into a nucleic acid of interest.

In some aspects, the invention relates to a method of isolating a nucleic acid having a predetermined sequence, the method comprising: providing at least one population of nucleic acid molecules; isolating a clonal population of nucleic acid molecules on a surface, determining the sequence of the clonal population of nucleic acid molecules, localizing the clonal population having a predetermined sequence, and amplifying the nucleic acid molecules having the predetermined sequence. In some embodiments, the separation step may be by dilution and the surface may be a flow cell.

In other aspects of the invention, a method of isolating nucleic acids having a predetermined sequence comprises providing a collection of nucleic acid molecules comprising error-free and error-containing nucleic acid molecules, tagging the nucleic acid molecules, optionally fragmenting the nucleic acid molecules, determining the sequence of the nucleic acid molecules, locating the error-free and error-containing nucleic acid molecules, and isolating the error-free nucleic acid molecules. In some embodiments, the separating step comprises one or more of: the method comprises the steps of ablating error-containing nucleic acid molecules, selectively amplifying error-free nucleic acid molecules, and/or immobilizing error-free nucleic acid molecules on a surface and separating error-free nucleic acid molecules from error-containing nucleic acid molecules. In some embodiments, the collection of nucleic acid molecules comprises at least 2 nucleic acid populations and each nucleic acid population can be immobilized on a different bead population. In some embodiments, the method further comprises sorting the different bead populations.

In some aspects of the invention, methods of sorting molecules having a predetermined sequence are provided. In some embodiments, the method comprises (a) providing a collection of nucleic acid molecules comprising at least 2 populations of nucleic acid molecules, each population of nucleic acid molecules having a unique target nucleic acid sequence with a 5 'end and a 3' end, (b) tagging the 5 'end and the 3' end of the target nucleic acid molecule with a pair of non-target oligonucleotide tag sequences, wherein the oligonucleotide tag sequences comprise unique nucleotide tags, (c) diluting the tagged target nucleic acids, (d) amplifying the tagged nucleic acids, (e) separating the amplified tagged nucleic acids into two collections, (f) subjecting the first collection comprising tagged target nucleic acid molecules to a sequencing reaction from both ends to obtain paired end reads; (g) subjecting the second pool comprising tagged target nucleic acid molecules to a ligation reaction to form circular nucleic acid molecules, thereby generating immediately adjacent tag pairs, (h) sequencing the tag pairs, (i) sorting the target nucleic acid molecules having the predetermined sequence according to their identity to their corresponding unique oligonucleotide tag pairs. In some embodiments, the tag pair may be amplified prior to sequencing. In some embodiments, the tag pairs may be excised prior to sequencing, e.g., using a restriction enzyme.

Brief description of the drawings

FIG. 1A shows steps I, II and III of a non-limiting exemplary method of making a clone, according to some embodiments. FIG. 1B shows steps IV and V of a non-limiting exemplary method of making a clone, according to some embodiments. FIG. 1C shows step VI of a non-limiting exemplary method of preparing clones, preparative recovery of correct clones, according to some embodiments. Asterisks indicate incorrect or undesired sequence sites.

FIG. 2A shows a non-limiting exemplary method of preparing an in vitro clonal sample according to some embodiments. FIG. 2B shows a non-limiting exemplary method of preparative in vitro clonal sequencing according to some embodiments. FIG. 2C shows a non-limiting exemplary method of in vitro clonal preparative isolation according to some embodiments.

FIG. 3 shows a non-limiting exemplary sample processing from a nucleic acid construct (C2G construct) to an in vitro cloning construct (IVC construct).

FIG. 4 shows a non-limiting exemplary flow chart of sequencing data analysis.

FIG. 5 shows a non-limiting exemplary alternative to plasmid-based barcoding.

FIG. 6 shows non-limiting exemplary parsing and scoring parsing.

FIG. 7A shows a non-limiting embodiment of isolating the source molecule. FIG. 7B shows a non-limiting embodiment of isolating the source molecule.

FIG. 8 shows a non-limiting exemplary isolation of a target nucleic acid using degenerate barcodes.

FIG. 9 shows a non-limiting exemplary isolation of nucleic acid clones from a collection of constructs using barcodes.

FIG. 10 shows a non-limiting exemplary isolation of nucleic acid clones from a collection of constructs using barcodes.

FIG. 11 shows a non-limiting exemplary embodiment of a bead-based recovery method.

FIG. 12 shows a non-limiting example of in vitro clonal integration with assembly.

FIG. 13A shows a non-limiting example of a reverse in vitro clone. FIG. 13B shows a non-limiting example of a reverse in vitro clone.

FIGS. 14A-C show a method according to a non-limiting embodiment of determining barcode pair information. FIG. 14A shows a pathway according to one embodiment through which barcoded ends of molecules are joined together by blunt end ligation of constructs to form a loop. Figure 14B shows a pathway according to another embodiment through which barcoded ends of molecules are joined together by blunt end ligation of constructs to form a loop. FIG. 14C shows a method in accordance with a non-limiting embodiment of linking barcodes to synthetic constructs. Figure 14D shows how constructs were sequenced in parallel and the isolated barcode pair can be used to identify the correct molecule for subsequent capture by amplification. X in the sequence indicates an error in the molecule.

FIG. 15 shows a non-limiting embodiment for determining barcode pair information.

Detailed Description

Techniques have been developed for de novo nucleic acid assembly whereby nucleic acids are prepared (e.g., chemically synthesized) and assembled to produce longer target nucleic acids of interest. For example, different multiplex assembly techniques are being developed to assemble oligonucleotides into larger synthetic nucleic acids. However, one limitation of currently available assembly techniques is the relatively high error rate. There is therefore a need to isolate nucleic acid constructs with a predetermined sequence and discard constructs with nucleic acid errors.

Aspects of the invention can be used to efficiently separate nucleic acid molecules from large numbers of nucleic acid fragments, and/or to reduce the number of steps required to produce large nucleic acid products, while reducing error rates. Aspects of the invention may also incorporate nucleic acid assembly processes to increase assembly fidelity, throughput, and/or efficiency, reduce costs, and/or shorten assembly time. In some embodiments, aspects of the invention can be automated and/or performed in a high throughput assembly environment to facilitate parallel production of many different target nucleic acid products. In some embodiments, nucleic acid constructs may be assembled using starting nucleic acids derived from one or more different sources (e.g., synthetic or natural polynucleotides, nucleic acid amplification products, nucleic acid degradation products, oligonucleotides, etc.). Aspects of the invention relate to the use of a high throughput platform for sequencing nucleic acids, such as assembled nucleic acid constructs, to identify high fidelity nucleic acids at lower cost. Such a platform has the advantage of being scalable, allowing for multiprocessing, producing a large number of sequence reads, having a fast turnaround time and being cost effective.

Some aspects of the invention relate to the preparation of construction oligonucleotides for high fidelity nucleic acid assembly. The present disclosure can be used to increase the production rate of nucleic acid assembly processes and/or reduce the number of steps or the amount of reagents used to generate correctly assembled nucleic acids. In certain embodiments, aspects of the invention can be used in the context of automated nucleic acid assembly to reduce the time, number of steps, amount of reagents, and other factors required for each correct nucleic acid assembly. These and other aspects of the invention may therefore be used to reduce the cost and time of one or more nucleic acid assembly processes.

The methods described herein can be used with any nucleic acid molecule, nucleic acid library, or nucleic acid collection. For example, the methods of the invention can be used to generate nucleic acid constructs, oligonucleotides or nucleic acid libraries having a predetermined sequence. In some embodiments, nucleic acid libraries can be obtained from commercial sources or can be designed and/or synthesized on a solid support (e.g., an array).

Analysis of

In some embodiments, the nucleic acid sequence of interest can be resolved into a set of construction oligonucleotides that together make up the nucleic acid sequence of interest. For example, in a first step, sequence information may be obtained. The sequence information may be the sequence of the nucleic acids of interest to be assembled. In some embodiments, the sequence may be accepted in the form of instructions from the client. In some embodiments, an acceptable sequence is a nucleic acid sequence (e.g., DNA or RNA). In some embodiments, it is acceptable that the sequence is a protein sequence. The sequence may be converted to a DNA sequence. For example, if the resulting sequence is an RNA sequence, T can be substituted for U to obtain the corresponding DNA sequence. If the resulting sequence is a protein sequence, it may be converted to a DNA sequence using appropriate amino acid codons.

In some embodiments, sequence information can be analyzed to determine an assembly strategy to generate a predetermined nucleic acid sequence of interest based on one or more of: the number of linkers, the length of the linkers, the sequence of the linkers, the number of fragments, the constants of the fragments, the sequence of the fragments to be assembled by sticky end ligation. In some embodiments, fragments can be assembled by sticky end ligation or by polymerase chain assembly.

In some embodiments, the assembly design is based on the length of the construction oligonucleotide and/or the number of linkers. For example, according to some embodiments, the length of the fragments may have an average length range of 98 to 104bp or 89 to 104 bp. In some embodiments, a design that produces a smaller number of fragments or linkers may be selected.

In some embodiments, sequence analysis may include scanning the linkers and selecting linkers having one or more of the following characteristics: each linker is 4 or more nucleotides in length, each linker differs from the other linkers by at least 2 nucleotides, and/or each linker differs from the other linkers by one or more nucleotides in the last 3 nucleotides of the linker sequence. The linkers may then be scored by the distance of the linkers in the linker sequence (also referred to herein as the Levenshtein distance). As used herein, the linker distance or Levenshtein distance corresponds to a measure of the difference between two sequences. Thus, the linker distance or Levenshtein distance between the first and second linker sequences corresponds to the number of single nucleotide changes required to change the first sequence to the second sequence. For example, a difference of 1 nucleotide in a 4 nucleotide sequence corresponds to a linker distance of 1, and a difference of 2 nucleotides in a 4 nucleotide sequence corresponds to a linker distance of 2. The joint distances may be averaged. In some embodiments, the joints are designed to have an average joint distance of 2 or more. In some embodiments, a design may be selected that results in a greater joint distance.

In some embodiments, all possible resolutions that satisfy the predetermined constraint are analyzed. If no effective resolution is found, the constraints can be relaxed to find a set of possible oligonucleotide sequences and linkers. For example, constraints on the length of oligonucleotides can be relaxed to include oligonucleotides having shorter or longer lengths.

In some embodiments, all possible resolutions that satisfy a predetermined constraint are ranked based on any metric provided herein. For example, each resolution may be ranked based on an average linker distance metric (as shown in fig. 6), GC content, complexity of the oligonucleotide sequence, and/or any other suitable metric.

In some embodiments, sequence analysis may include scanning for the presence of one or more interfering sequence features that are known or predicted to interfere with oligonucleotide synthesis, amplification, or assembly. For example, the interfering sequence structure may be a sequence with low GC content (e.g., less than 30% GC, less than 20% GC, less than 10% GC, etc.) over a length of at least 10 bases (e.g., 10-20 bases, 20-50 bases, 50-100 bases, or more than 100 bases), or a sequence that may form a secondary structure or a stem-loop structure.

In some embodiments, synthetic construction oligonucleotides (e.g., sequence, size, and number) may be designed for assembly after the construct quantification and resolution steps. Standard DNA synthesis chemistry (e.g., phosphoramidite methods) can be used to generate synthetic oligonucleotides. Synthetic oligonucleotides can be synthesized on a solid support (e.g., a microarray) using any suitable technique known in the art. The oligonucleotides may be eluted from the microarray prior to amplification or amplified on the microarray.

As used herein, an oligonucleotide may be a nucleic acid molecule comprising at least two covalently bound nucleotide residues. In some embodiments, the oligonucleotide may be 10-1000 nucleotides in length. For example, the oligonucleotide may be 10-500 nucleotides in length, or 500-1000 nucleotides in length. In some embodiments, the oligonucleotide may be about 20 to about 300 nucleotides in length (e.g., about 30 to 250, 40 to 220, 50 to 200, 60 to 180, or about 65 or about 150 nucleotides), about 100 to about 200 nucleotides, about 200 to about 300 nucleotides, about 300 to about 400 nucleotides, or about 400 to about 500 nucleotides. However, shorter or longer oligonucleotides may be used. The oligonucleotide may be a double-stranded or single-stranded nucleic acid. The terms "nucleic acid," "polynucleotide," "oligonucleotide" are used interchangeably herein and refer to a naturally occurring or synthetic polymeric form of nucleotides. The oligonucleotides and nucleic acid molecules of the invention may be formed from naturally occurring nucleotides, for example, to form deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules. Alternatively, the naturally occurring oligonucleotide may comprise a structural modification that alters its properties, such as a Peptide Nucleic Acid (PNA) or a Locked Nucleic Acid (LNA). Solid phase synthesis of oligonucleotides and nucleic acid molecules having naturally occurring bases or artificial bases is well known in the art. It is to be understood that the term encompasses equivalents, analogs of RNA or DNA generated from nucleotide analogs, and single-or double-stranded polynucleotides as applied to the embodiments to be described. Nucleotides useful in the invention comprise, for example, naturally occurring nucleotides (e.g., ribonucleotides or deoxyribonucleotides), or natural or synthetic modifications of nucleotides, or artificial bases. The term monomer as used herein refers to a member of a small group of molecules that are and can be joined together to form an oligomer, a polymer, or a compound made up of two or more members. The particular order of the monomers in the polymer is referred to herein as the "sequence" of the polymer. The monomer groups include, but are not limited to, for example, the common L-amino acid group, the D-amino acid group, the synthetic and/or natural amino acid group, the nucleotide group, and the pentose and hexose group. Aspects of the invention described herein relate primarily to the preparation of oligonucleotides, but are readily applicable to the preparation of other polymers such as peptides or polypeptides, polysaccharides, phospholipids, heteropolymers, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates or any other polymer.

Nucleosides are typically linked by phosphodiester linkages. When a nucleic acid is represented by a letter sequence, it is understood that the nucleotides are in 5 'to 3' order from left to right. According to IUPAC notation, "A" represents deoxyadenosine, "C" represents deoxycytidine, "G" represents deoxyguanosine, "T" represents deoxythymidine, "U" represents ribonucleoside, uridine. In addition, there are also letters used when more than one nucleotide may be present at that position: "W" (i.e., a weak bond) represents A or T, "S" (a strong bond) represents G or C, "M" (for amino) represents A or C, "K" (for ketone) represents G or T, "R" (for purine) represents A or G, "Y" (for pyrimidine) represents C or T, "B" represents C, G or T, "D" represents A, G or T, "H" represents A, C or T, "V" represents A, C or G and "N" represents any base A, C, G or T (U). It is understood that the nucleic acid sequence is not limited to the 4 natural deoxynucleotides, but may also include ribonucleosides or non-natural nucleotides.

In some embodiments, the methods and devices provided herein can use oligonucleotides immobilized on a surface or substrate (e.g., a support-bound oligonucleotide), wherein the 3 'or 5' end of the oligonucleotide is bound to the surface. Support-bound oligonucleotides include, for example, oligonucleotides complementary to the construction oligonucleotides, anchor oligonucleotides, and/or spacer oligonucleotides. The terms "support", "substrate" and "surface" as used herein are used interchangeably and refer to a porous or non-porous solvent-insoluble material on which polymers, such as nucleic acids, are synthesized or immobilized. As used herein, "porous" means that the material contains pores of substantially uniform diameter (e.g., in the nm range). Porous materials include paper, synthetic filters, polymer matrices, and the like. In such porous materials, the reaction may be carried out in the pores or matrix. The support can have any of a number of shapes, such as pins, strips, plates, flat discs, rods, bends, cylindrical structures, particles (including beads, nanoparticles), and the like. The support can have a variable width. The support may be hydrophilic or capable of exhibiting hydrophilicity. The support may comprise inorganic powders such as silica, magnesium sulfate, and alumina; natural polymeric materials, in particular cellulosic materials and cellulose derived materials, for example paper comprising fibres, such as filter paper, chromatography paper and the like; synthetic or modified naturally occurring polymers such as nitrocellulose, cellulose acetate, poly (vinyl chloride), polyacrylamide, cross-linked dextran, agarose, polyacrylate, polyethylene, polypropylene, poly (4-methylbutene), polystyrene, polymethacrylate, poly (ethylene terephthalate), nylon, poly (vinyl butyrate), polyvinylidene fluoride (PVDF) membrane, glass, controlled pore glass, magnetic controlled pore glass, ceramics, metals, and the like; either alone or in combination with other materials. In some embodiments, the oligonucleotides are synthesized on an array format. For example, single stranded oligonucleotides are synthesized in situ on a common support, wherein each oligonucleotide is synthesized on a separate or discrete feature (or spot) of the substrate. In some embodiments, single stranded oligonucleotides may be bound to a surface or feature of the support. The term "array" as used herein refers to an arrangement of discrete features for storing, amplifying and releasing oligonucleotides or complementary oligonucleotides for further reaction. In some embodiments, the support or array is addressable: the support comprises two or more discrete addressable features at specific predetermined locations (i.e., "addresses") on the support. Thus, each oligonucleotide molecule on the array is located at a known and defined position on the support. Each oligonucleotide sequence can be determined from its site on the support.

In some embodiments, the oligonucleotides are attached, spotted, immobilized, surface bound, supported, or synthesized on a surface or discrete features of an array. The oligonucleotides may be covalently attached to the surface or deposited on the surface. Arrays can be constructed, custom purchased, or purchased from commercial vendors (e.g., Agilent, Affymetrix, Nimblegen). Various construction methods are well known in the art, such as maskless array synthesizers, light guide methods using masks, flow channel methods, spotting methods, and the like. In some embodiments, maskless arrays can be used to construct and/or select oligonucleotidesThe synthesizer (MAS) is synthesized on a solid support. Maskless array synthesizers are described, for example, in PCT application No. WO99/42813 and corresponding U.S. patent No. 6,375,903. Other examples are known maskless devices that can make custom DNA microarrays in which each feature in the array has a single-stranded DNA molecule of a desired sequence. Other methods of synthesizing oligonucleotides include, for example, a photo-orientation method using a mask, a flow channel method, a spotting method, a pin-based method, and a method using a plurality of supports. Light-directing method using masks for synthesizing oligonucleotides (e.g., VLSIPS^TMMethods) are described in U.S. Pat. nos. 5,143,854, 5,510,270 and 5,527,681. These methods involve activating a predetermined area on a solid support and then contacting the support with a pre-selected monomer solution. The selected areas can be activated by irradiation of a light source through a mask, most often in accordance with photolithographic techniques used in the fabrication of integrated circuits. Other areas of the support remain inactive because the irradiation is blocked by the mask and it remains chemically protected. Thus, the light pattern defines which region on the support reacts with a given monomer. Different arrays of polymers are produced on the support by repeatedly activating different sets of predetermined regions and contacting different monomer solutions to the support. The method can also be effected by using a photoresist that is compatible with the growing surface binding molecules and the synthetic chemistry involved. Other steps can optionally be used, such as washing the unreacted monomer solution from the support. Other applicable methods include mechanical techniques such as those described in U.S. Pat. No. 5,384,261. Other methods that can be used to synthesize oligonucleotides on a single support are described, for example, in U.S. Pat. No. 5,384,261. For example, reagents can be delivered to the support by (1) flowing in a channel defined over a predetermined area, or (2) "spotting" on a predetermined area. Other methods and combinations of spotting and flow can also be used. In each instance, certain activated regions on the support are mechanically separated from other regions when the monomer solution is delivered to multiple reaction sites. Flow channel methods include, for example, microfluidic systems to control oligonucleotides on solid supportsAnd (4) synthesizing acid. For example, different polymer sequences may be synthesized on selected regions of a solid support by forming flow channels over the surface of the support through which appropriate reagents flow or in which appropriate reagents are placed. Spotting methods for preparing oligonucleotides on solid supports involve the delivery of relatively small amounts of reactants by direct placement in selected areas. In some steps, the monolithic support surface can be sprayed or coated with a solution if it is more effective to do so. Accurately measured aliquots of the monomer solution may be placed drop-wise by a dispenser that moves from one area to another. Pin-type methods for synthesizing oligonucleotides on solid supports are described, for example, in U.S. Pat. No. 5,288,514. Pin-type methods utilize supports having multiple pins or other extensions. The pin types are each inserted simultaneously into a single reagent container of the tray. 96 pin type arrays typically utilize a 96 well tray, such as a 96 well microtiter dish. Each tray is filled with a specific reagent coupled in a specific chemical reaction on a single pin. Thus, the trays will often contain different reagents. Multiple chemical coupling steps can be performed simultaneously, since the chemical reactions are optimized such that each reaction can be performed under a relatively similar set of reaction conditions.

In another embodiment, multiple oligonucleotides may be synthesized or immobilized on multiple supports. One example is described in, for example, U.S. patent nos. 5,770,358; 5,639,603, respectively; and 5,541,061. To synthesize molecules, such as oligonucleotides, on the beads, a large number of beads are suspended in a suitable carrier (e.g., water) in a container. Beads are provided with optional spacer molecules having active sites for their complexation, optionally protecting groups. In each step of the synthesis, the beads are separated for coupling into various containers. After deprotection of the nascent oligonucleotide chain, different monomer solutions are added to each vessel so that the same nucleotide addition reaction occurs on all beads in a given vessel. The beads are then washed with excess reagent, collected into a single vessel, mixed and redistributed to another vessel in preparation for the next round of synthesis. It should be noted that due to the large number of beads initially utilized, a large number of beads will likewise be randomly distributed within the container, each having a unique oligonucleotide sequence synthesized on its surface after multiple rounds of random base addition. Individual beads can be tagged with a sequence that is unique to the double-stranded oligonucleotide on it, allowing identification in the application.

The pre-synthesized oligonucleotide and/or polynucleotide sequences may be attached to a support or synthesized in situ using the following methods: light directing processes, flow channel and spotting processes, ink jet processes, pin processes and bead based processes are shown in the following references: McGall et al, (1996) proc.natl.acad.sci.u.s.a.93: 13555, preparing a mixture; synthesis of DNA Arrays In Genetic Engineering operations (Synthetic DNA Arrays In Genetic Engineering), volume 20: 111, pleinan Press (Plenum Press) (1998); duggan et al, (1999) nat. genet.s21: 10; microarray: produced and used In microarray bioinformatics (microarray: manufacturing and Using the same In microarray bioinformatics), Cambridge University Press (Cambridge University Press), 2003; U.S. patent application publication nos. 2003/0068633 and 2002/0081582; U.S. patent nos. 6,833,450, 6,830,890, 6,824,866, 6,800,439, 6,375,903 and 5,700,637; and PCT publication nos. WO 04/031399, WO 04/031351, WO 04/029586, WO 03/100012, WO 03/066212, WO 03/065038, WO 03/064699, WO 03/064027, WO03/064026, WO 03/046223, WO 03/040410 and WO 02/24597; the disclosure is incorporated by reference herein in its entirety for all purposes. In some embodiments, the presynthesized oligonucleotides are attached to a support or synthesized using a spotting method, wherein the monomer solution is deposited dropwise through a dispenser (e.g., an ink jet) that moves from one region to another. In some embodiments, oligonucleotides are spotted on the support using, for example, a mechanical wave driven dispenser.

In some embodiments, the assembled nucleic acid fragments or constructs (also referred to herein as nucleic acids of interest) can each be about 100 nucleotides to about 1000 nucleotides in length (e.g., about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900). However, longer (e.g., about 2500 or more nucleotides in length, about 5000 or more nucleotides in length, about 7500 or more nucleotides in length, about 10000 or more nucleotides in length, etc.) or shorter nucleic acid fragments can be assembled using assembly techniques (e.g., shotgun assembly into plasmid vectors). It is understood that the size of each nucleic acid fragment is independent of the size of the other nucleic acid fragments added to the assembly. However, in some embodiments, the nucleic acid fragments may be about the same size.

Aspects of the invention relate to methods and compositions for selectively isolating nucleic acid constructs having a predetermined sequence of interest. The term "predetermined sequence" as used herein refers to a sequence of a polymer that is known and selected prior to synthesis or assembly of the polymer. In particular, aspects of the invention described herein relate primarily to the preparation of nucleic acid molecules, the sequence of oligonucleotides or polynucleotides that are known and selected prior to synthesis or assembly of the nucleic acid molecules. In some embodiments of the technology provided herein, immobilized oligonucleotides or polynucleotides are used as a source of material. In various embodiments, the methods described herein use a plurality of construction oligonucleotides, each oligonucleotide having a target sequence determined based on the sequence of the final nucleic acid construct to be synthesized (also referred to herein as the nucleic acid of interest). In one embodiment, the oligonucleotide is a short nucleic acid molecule. For example, the oligonucleotide may be 10 to about 300 nucleotides, 20 to about 400 nucleotides, 30 to about 500 nucleotides, 40 to about 600 nucleotides, or more than about 600 nucleotides in length. However, shorter or longer oligonucleotides may be used. Oligonucleotides can be designed to have different lengths. In some embodiments, the sequence of a polynucleotide construct can be divided into a variety of shorter sequences (e.g., construction oligonucleotides) that can be synthesized in parallel and assembled into a single or multiple desired polynucleotide constructs using the methods described herein. Prior to processing (e.g., tagging, diluting, amplifying, sequencing, separating, assembling, etc.), nucleic acids can be collected from one or more arrays, such as by constructing oligonucleotides, to form a library or collection of nucleic acids.

According to some aspects of the invention, each nucleic acid sequence to be assembled (also referred to herein as a nucleic acid source molecule) can include an internal predetermined target sequence having a 5 'end and a 3' end and additional flanking sequences at the 5 'end and/or the 3' end of the internal target sequence. In some embodiments, an internal target sequence or a nucleic acid comprising the internal target sequence and additional 5 'and 3' flanking sequences can be synthesized onto a solid support as described herein.

In some embodiments, the synthetic nucleic acid sequence comprises an internal target sequence and non-target sequences upstream and downstream of the target sequence. In some embodiments, the non-target sequence may include a sequence id (seqid) for identifying similar target sequences at the 3 'end (downstream) and 5' end (upstream) of the target sequence and a sequencing handle (H) for multiplex sample preparation at the 3 'end and 5' end of the target sequence. The sequencing handles may be located at the 3 'end and the 5' end of the sequence ID. In some embodiments, the sequence ID is 10 nucleotides in length. In some embodiments, sequencing handle H is 20 nucleotides in length. However, shorter or longer sequence IDs and/or sequencing handles may be used. In some embodiments, additional sequences, such as oligonucleotide tag sequences, may be used to synthesize the nucleic acid sequence. For example, the nucleic acid sequences may be designed such that they comprise an oligonucleotide tag sequence selected from a library of oligonucleotide tag sequences as described herein. In some embodiments, the nucleic acid sequence may be designed to have an oligonucleotide tag sequence that comprises a sequence in common among a set of nucleic acid constructs. The term "common sequence" means that the sequences are identical. In some embodiments, the common sequence may be a universal sequence. In other embodiments, the 5 'oligonucleotide tag sequence is designed to have a common sequence at its 3' end and the 3 'oligonucleotide tag sequence is designed to have a common sequence at its 5' end. For example, a nucleic acid can be designed to have a common sequence at the 3 'end of its 5' oligonucleotide tag and at the 5 'end of the 3' oligonucleotide tag. Libraries of oligonucleotide tag sequences can be used for nucleic acid constructs to be assembled from a single array. In other embodiments, the library of oligonucleotide tags may be reused for different constructs generated from different arrays. In some embodiments, a library of oligonucleotide tag sequences can be designed to be universal. In some embodiments, the nucleic acid or oligonucleotide tag is designed to have additional sequences. The additional sequences may include any nucleotide sequence suitable for sequencing, amplifying, isolating, or assembling the nucleic acids in the collection.

Preparative In Vitro Cloning (IVC) method

Provided herein are preparative in vitro cloning methods or strategies for de novo synthesis of high fidelity nucleic acids. In some embodiments, the in vitro cloning methods can use oligonucleotide tags. In other embodiments, the in vitro cloning method does not require the use of oligonucleotide tags.

In some embodiments, the methods described herein allow for the cloning of a nucleic acid sequence having a desired or predetermined sequence from a collection of nucleic acid molecules. In some embodiments, the method can include analyzing the sequence of a target nucleic acid for parallel preparative cloning of a plurality of target nucleic acids. For example, the methods described herein can include quality control steps and/or quality control reads to identify nucleic acid molecules with the correct sequence. FIGS. 1A-C show an exemplary method for isolating and cloning a nucleic acid molecule having a predetermined sequence. In some embodiments, the nucleic acid may be synthesized or assembled onto the support first. For example, nucleic acid molecules can be assembled in 96-well plates with one construct per well. In some embodiments, each nucleic acid construct (C)₁To C_NFIGS. 1A-C) have different nucleotide sequences. For example, the nucleic acid construct may be a non-homologous nucleic acid sequence or a nucleic acid sequence with a degree of homology. In other embodiments, multiple nucleic acid molecules having a predetermined sequence, such as C, can be deposited on different locations or wells of a solid support₁To C_N. In some embodiments, the length limitation of a nucleic acid construct may depend on the efficiency of sequencing the 5 'end and the 3' end of the full-length target nucleic acid by high-throughput paired-end sequencing. One skilled in the art will appreciate that the methods described herein can bypass the need for cloning by transforming cells with the nucleic acid construct in a propagation vector (i.e., in vivo cloning). In addition, the methods described herein eliminate the need to separately amplify the candidate construct prior to identifying the target nucleic acid having the desired sequence.

One skilled in the art will appreciate that after oligonucleotide assembly, the assembly product may contain a collection of sequences that contain both correct and incorrect assembly products. For example, referring to FIG. 1A, wells of plate (nucleic acid construct C)₁To C_N) It may be a mixture of nucleic acid molecules with correct or incorrect sequences (the wrong sequence site is indicated by an asterisk). Errors may be caused by sequence errors introduced during oligonucleotide synthesis, or during assembly of oligonucleotides into longer nucleic acids. In some cases, up to 90% of the nucleic acid sequence may be undesired sequences. Provided herein are devices and methods for selectively separating a correct nucleic acid sequence from an incorrect nucleic acid sequence. The correct sequence can be isolated by selective separation from other incorrect sequences, e.g., by selective movement or transfer of the desired assembled polynucleotide of the predetermined sequence to a different feature of the support, or to another plate. Alternatively, polynucleotides with erroneous sequences can be selectively removed from features comprising the polynucleotide of interest. According to some methods of the invention, the assembled nucleic acid molecules may first be diluted in a solid support to obtain a standardized population of nucleic acid molecules. The term "normalizing" or "normalized collection" as used herein means that a collection of nucleic acids has been treated to reduce the relative change in abundance between members of the nucleic acid molecules in the collection to no more than about 1000-fold, no more than about 100-fold, no more than about 10-fold, no more than about 5-fold, no more than about 4-fold, no more than about 3-fold, or no more than about 2-fold. In some embodiments, the nucleic acid molecule is normalized by dilution. For example, the nucleic acid molecules can be normalized such that the number of nucleic acid molecules is about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 1000, or higher. In some embodiments, the complexity of the collection can be reduced by normalizing each population of nucleic acid molecules by limited dilution prior to collection of the nucleic acid molecules. In some embodiments, to ensure that at least one copy of the target nucleic acid is present in the collection, the dilution is limited to providing more than one nucleic acid molecule. In some embodiments, the oligonucleotide may be diluted serially. In some embodiments, the device (e.g., array or microwell plate,e.g., 96-well plates) may incorporate serial dilution functionality. In some embodiments, the assembly product can be serially diluted to generate a standardized population of nucleic acids. The concentration and number of molecules can be assessed prior to the dilution step and the dilution ratio calculated to produce a normalized population. In exemplary embodiments, the assembled product is diluted by a factor of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 10, at least 20, at least 50, at least 100, at least 1000, and the like. In some embodiments, prior to sequencing, the target nucleic acid sequence may be diluted and placed, for example, in a different well or on a different location of a solid support or on a different support.

In some embodiments, the normalized populations of nucleic acid molecules can be pooled to generate a collection of nucleic acid molecules having different predetermined sequences. In some embodiments, each nucleic acid molecule in the collection can be at a relatively low complexity. In other embodiments, normalization of nucleic acid molecules can be performed after mixing different populations of nucleic acid molecules present at high concentrations.

In other embodiments, the method of the present invention comprises the following steps as shown in fig. 2A: (a) providing a collection of different nucleic acid constructs (also referred to herein as source molecules); (b) providing a pool of oligonucleotide tags, each oligonucleotide tag comprising a unique nucleotide tag sequence or barcode; (c) ligating oligonucleotide tags (K and L) to each source molecule in a collection of nucleic acid molecules at the 5 'end and the 3' end such that substantially all of the different molecules in the collection have different pairs of oligonucleotide tags (K, L) attached thereto to thereby ligate the barcode to a particular source molecule, and (d) diluting the tagged nucleic acid sequences; (e) obtaining paired-end reads of each nucleic acid molecule; and (f) sorting nucleic acid molecules having the desired predetermined sequence according to the identity of the barcode. The term "barcode" as used herein refers to a unique oligonucleotide tag sequence that allows for the identification of a corresponding nucleic acid sequence. By designing libraries or libraries of barcodes to form a sufficiently large barcode library relative to the number of nucleic acid molecules, each different nucleic acid molecule can have a unique barcode pair. In some embodiments, the library of barcodes comprises a plurality of 5 'end barcodes and a plurality of 3' end barcodes. Each 5 'terminal barcode of the library can be designed to have a 3' terminal or internal sequence in common with each member of the library. Each 3 'terminal barcode of the library can be designed to have a 5' terminal or internal sequence in common with each member of the library.

In some embodiments, the method further comprises using Nextera^TMTagmentation to digest tagged source molecules and usesOr higher throughput sequencing of a new generation sequencing platform. Nextera^TMThe tagged paired reads typically result in one sequence with the oligonucleotide tag sequence for recognition, and a sequence within the target region of the other construct (as shown in FIG. 2C). With high throughput sequencing, sufficient coverage can be generated to reconstruct the consensus sequence of each tag pair construct and determine if the sequence is correct (i.e., error-free).

In some embodiments, nucleic acid molecules can be pooled from one or more solid supports for multiplexed processing. The nucleic acid molecules may be diluted to maintain a manageable number of clones per target nucleic acid molecule. Each nucleic acid molecule can be tagged by adding a unique barcode or unique pair of barcodes to each end of the molecule. Diluting the nucleic acid molecules prior to ligation of the oligonucleotide tags may allow for a reduction in the complexity of the collection of nucleic acid molecules, thereby allowing for the use of a reduced complexity barcode library. The tagged molecules may then be amplified. In some embodiments, the oligonucleotide tag sequence may include a primer binding site for amplification (fig. 2C). In some embodiments, oligonucleotide tag sequences can be used as primer binding sites. The amplified tagged molecules can be tagged and subjected to pair-wise read sequencing to associate the barcode with the desired target sequence. The barcode can be used as a primer to recover a sequence clone having a desired sequence. Amplification methods are well known in the art. Examples of enzymes with polymerase activity that can be used for amplification by PCR are NA polymerase (klenow fragment, T4 DNA polymerase), thermostable DNA polymerases from a variety of thermostable bacteria (Taq, ven, Pfu or Tfl DNA polymerases) and their genetically modified derivatives (Taq gold, VENTexo, Pfu exo) or KOD Hifi DNA polymerase. In some embodiments, amplification by chimeric PCR can reduce the signal-to-noise ratio associated with barcodes.

In other embodiments, nucleic acid molecules can be pooled from one or more arrays for multiplex processing. As described herein, a nucleic acid molecule can be designed such that it comprises barcodes at the 5 'and 3' ends. In some embodiments, the barcode has a sequence in common within a construct and between a set of constructs. For example, barcodes may be generic to each construct assembled from a single array. In some embodiments, the barcodes may have a common linker sequence or a common primer binding site sequence.

In some embodiments, barcodes may be added to the nucleic acid molecules and the tagged nucleic acid molecules may be diluted prior to being amplified. The amplified tagged molecules can be tagged and sequenced to associate a barcode pair with each nucleic acid molecule. In some embodiments, one read of each read pair is used for sequencing the barcoded ends. Read pairs that do not contain any barcodes may be filtered out. The sequencing error rate can be removed by consensus (trapping). For example, barcodes can be used as primers to isolate nucleic acid molecules having a desired sequence.

According to some methods of the invention, a nucleic acid sequence (a building oligonucleotide, an assembly intermediate, or an assembled nucleic acid of interest) may be first diluted to obtain a clonal population of target polynucleotides (i.e., a population containing a single target polynucleotide sequence). As used herein, "cloned nucleic acid" or "clonal population" or "cloned polynucleotide" are used interchangeably and refer to a population of clonal molecules of nucleic acid, i.e., nucleic acids that are substantially or completely identical to each other. Thus, dilution-based methods provide populations of nucleic acid molecules that are substantially identical or identical to each other. In some embodiments, the polynucleotide may be serially diluted. The concentration and quantity of molecules can be assessed prior to the dilution step, so that dilution rates can be calculated to generate clonal populations.

In some embodiments, Next Generation Sequencing (NGS) dot locations or microfluidic channel locations can be used as nucleic acid construct markers that eliminate the need for designing construct-specific barcodes.

In some embodiments, when using a flow cell having multiple flow cells (e.g.,2000) with NGS, an average of 1 clone per flow cell for each gene is possible. Limited dilution, as determined by poisson distribution, will result in a single hit (e.g., one clone per well). Poisson statistics show that if the average clone number for each gene is 1 per flow cell, then a flow cell of approximately 1/3 will have 0 clones, a flow cell of 1/3 will have 1 clone and a flow cell of 1/3 will have 2 clones. Thus, if the error rate requires N clones to produce a perfect or error-free full-length construct, 3 x N flow cells would be required to get a high probability that at least one flow cell would contain a clonal representation of a perfect construct. For example, if N ═ 4, then 12 flow cells would be required. In some embodiments, after sequencing clones within the flow cells, a means of collecting the effluent of each flow cell into separate wells may be provided. Sequencing data can then be used to identify collection wells containing nucleic acids having a predetermined sequence. After determining which nucleic acids having the predetermined sequence are in those collection wells, primers specific for the nucleic acids having the predetermined sequence can be used to amplify the nucleic acids having the predetermined sequence from their appropriate wells. In such embodiments, the primer may be complementary to the nucleic acid sequence of interest and/or the oligonucleotide tag.

Tag oligonucleotide

In some embodiments, a pair of tag oligonucleotide sequences can be used to tag the 5 'end and the 3' end of each nucleic acid molecule within a collection. In some embodiments, the tag oligonucleotide sequence may consist of a common DNA primer region and a unique "barcode" region, such as a specific nucleotide sequence. In some embodiments, the number of tag nucleotide sequences may be greater than the number of molecules per construct (i.e., 10-1000 molecules in dilution).

In some embodiments, the barcode sequence can also function as a primer binding site to amplify a barcoded nucleic acid molecule or to isolate a nucleic acid molecule having a desired predetermined sequence. In such embodiments, the terms barcode and oligonucleotide tag are used interchangeably. In such embodiments, the terms "barcoded nucleic acid" and "tagged nucleic acid" are used interchangeably. It is understood that the oligonucleotide tag may have any suitable length and composition. In some embodiments, oligonucleotide tags can be designed to (a) generate a sufficiently large barcode library to allow tagging of each end of each nucleic acid molecule with a unique barcode; and (b) minimize cross-hybridization between different barcodes. In some embodiments, the nucleotide sequence of each barcode differs sufficiently from any other barcode in the library that no member of the barcode library can form a dimer under the reaction conditions used, such as hybridization conditions.

In some embodiments, the barcode sequence may be 6bp, 7bp, 8bp, 9bp, 10bp, 12bp, 13bp, 14bp, 15bp, 16bp, 17bp, 18bp, 19bp, 20bp, 21bp, 22bp, 23bp, 24bp, 25bp, 26bp, 27bp, 28bp, 29bp, 30bp, or more than 30bp in length. In some embodiments, the 5 'end barcode sequence and the 3' end barcode sequence may be different in length. For example, the 5 'barcode may be 14 nucleotides in length and the 3' barcode may be 20 nucleotides in length. In some embodiments, the length of the barcode may be selected to minimize space reduction of the barcode, maximize barcode space at the 3' end, allow correction of barcode errors, and/or minimize variation between barcode melting temperatures for primer capability (primality) considerations. For example, the melting temperatures of barcodes within a set may be within 10 ℃ of each other, within 5 ℃ of each other, or within 2 ℃ of each other.

Each barcode sequence may comprise a fully degenerate sequence, a partially degenerate sequence, or a non-degenerate sequence.

For example, nucleotide tags of 6bp, 7bp, 8bp or longer may be used. In some embodiments, a degenerate sequence of NNNNNNNN (8 degenerate bases, where each N can be any natural or non-natural nucleotide) can be used and generate 65536 unique barcodes. In some embodiments, the length of the nucleotide tags can be selected to limit the number of tag pairs having a common tag sequence for each nucleic acid construct.

It will be appreciated by those skilled in the art that fully degenerate sequences may produce a high number of different barcodes, but may also produce higher variations in the melting temperature Tm of the primers. The melting temperature is the temperature at which half of a population of double-stranded nucleic acid molecules dissociates into single strands. Formulas for calculating the Tm of nucleic acids are well known in the art. For example, when a nucleic acid is in an aqueous solution of 1M NaCl, the Tm value can be simply estimated by calculation from the formula Tm of 81.5 ± 0.41 (% G + C). In some embodiments, the barcode sequence is an encoded barcode and may comprise a partially degenerate sequence in combination with fixed or constant nucleotides. In some embodiments, the barcode may include one or more of the following: (a) a degenerate base N at the 3' end; (b) one or more C at the 5' end (to limit Tm); (c) including W, D, H, S, B, V and the extension of M.

In some embodiments, the barcode is an encoded barcode and may include, but is not limited to, a barcode library having the following sequence:

bar code 1: CCWSWDHSHDBVHDNNMM. The 20 base barcodes had the same barcode degenerate space as 13N.

The bar code 2: CCSWHDSDHVBDHNNNNMM. Compared to barcode 1, the 21 base barcode has some degenerate bases that are switched in position. It should be noted that the primers can be distinguished between barcode 1 and barcode 2.

In some embodiments, barcode sequences can be designed, analyzed, and ranked to produce a list of ranked nucleotide tags, which are enriched for perfect sequence and primer performance. It will be appreciated that the encoded barcodes provide a means for generating primers with a higher Tm range.

In some embodiments, a tag oligonucleotide sequence or barcode may be attached to each nucleic acid molecule to form a nucleic acid molecule comprising the tag oligonucleotide sequence at its 5 'and 3' ends. In some embodiments, a ligase may be used to ligate the tag oligonucleotide sequence or barcode to the blunt-ended nucleic acid molecule. For example, the ligase may be a T7 ligase or any other ligase capable of ligating the tag oligonucleotide sequence to the nucleic acid molecule. Ligation may be performed under conditions suitable to avoid concatamerization of the nucleic acid constructs. In other embodiments, the nucleic acid molecule is designed to have sequences common to or complementary to the tag oligonucleotide sequence at its 5 'and 3' ends. In some embodiments, the tag oligonucleotide sequence and the nucleic acid molecule having a common sequence can be linked by polymerase chain reaction, such as an aptamer (adaptor). As shown in FIG. 2A, barcodes can be attached at the 5 'end and 3' end of sequencing handle H (A and B), which flank the internal target sequence. In some embodiments, each source molecule synthesized on the first solid support has a common pair of sequencing handles at its 5 'and 3' ends. For example, oligonucleotides synthesized on a first solid support have a first pair of sequencing handles (A1, B1), while oligonucleotides synthesized on a second solid support have a second pair of sequencing handles (A2, B2), and so on.

In other embodiments, the barcode may be introduced by ligation to the 5 'and 3' ends of the nucleic acid molecule without addition of the sequence identifier SeqID and/or the sequencing handle H. Thus, the construct primer is still intact and can function as a sequence identifier. This approach may have the advantage of using nucleic acid constructs synthesized onto the array with internal target sequences and primer regions at the 5 'and 3' ends of the target sequences, and with greater control to normalize the constructs. In some embodiments, barcodes can be introduced using the plasmid-based method shown in fig. 4, comprising the steps of: (1) providing a barcoded vector (e.g., pUC19 vector), (2) providing a core assembly construct or oligonucleotide, (3) phosphorylating the nucleic acid construct; (4) ligating the barcoded vector and the core assembly construct, and (5) pooling the ligation products; and (6) diluting and/or amplifying the ligation product. For example, the linearized vector includes 5 'and 3' flanking regions. In some embodiments, the flanking region can be designed such that it has an external barcode and an internal sequence adaptor. For example, the flanking regions may include barcodes, tagged adapters, and M13 sequences. It is to be understood that this alternative barcoding scheme need not be plasmid-based and that any linear nucleic acid fragment having barcodes at its 5 'and 3' ends may be used.

FIG. 3 shows the workflow of the above-described method of tagging a population of target nucleic acid sequences with oligonucleotide tags, sequencing molecules to obtain oligonucleotide tags and internal target sequencing information, and recovering the desired tagged construct sequences. The workflow/inventive process can be simplified as follows: the target molecule group (a) > tag (B) > sequencing (C) > target nucleic acid sequence (D) required for recovery.

In other embodiments, and with reference to fig. 12A-B, a nucleic acid construct can be assembled from a variety of internal target sequence fragments and unique barcode sequences. Unique barcode sequences can be designed to assemble with the target sequence at both the 5 'end and the 3' end of the internal target sequence to generate a population of molecules with unique flanking barcode sequences and an internal target region of interest. In some embodiments, the 5 'terminal internal target sequence fragment is designed to have the sequence identifier SeqID and/or the sequencing handle H at its 5' end and the 3 'terminal internal target sequence fragment is designed to have the sequence identifier SeqID and/or the sequencing handle H at its 3' end. The advantage of this approach is the integration of the in vitro cloning method (IVC method) with the assembly method (also referred to herein as the C2G assembly method). As shown in FIGS. 12A-B, each assembled molecule with an internal target of interest has a different sequence pair (K)_i，L_i) Such as (K)_i1，L_i1)、(K_i2，L_i2) Etc. to distinguish them from other molecules in the collection of nucleic acid constructs. In some embodiments, multiple constructs (e.g., C) with different internal target sequences of interest can be mixed in a set_A1、C_B1And C_C1) (FIG. 12C). The different constructs can be diluted, amplified and sequenced as described herein and shown in fig. 12D. Nucleic acid molecules having a desired sequence can be sorted according to identity of the corresponding unique barcode pair.

One skilled in the art will appreciate the advantage of the foregoing method in that the constructs are not tagged, since the core population of molecules is already substantially equivalent to processing point B in the above described workflow. The workflow may then be described as follows: the unique target molecule population (a') > sequencing (C) > recovering the desired target nucleic acid sequence (D).

Sequencing

In some embodiments, a target nucleic acid sequence or a copy of a target nucleic acid sequence, some of which contain one or more sequence errors, can be isolated from a collection of nucleic acid sequences. As used herein, a copy of a nucleic acid sequence of interest refers to a copy using a template-dependent method such as PCR. In some embodiments, sequencing of a target nucleic acid sequence can be performed using sequencing of individual molecules (e.g., single molecule sequencing) or sequencing of an amplified population of target nucleic acid sequences (e.g., polymerase clone sequencing). In some embodiments, the collection of nucleic acid molecules is subjected to a high throughput paired-end sequencing reaction, e.g., using

(lllumina) or any suitable next generation sequencing system (NGS).

In some embodiments, a common primer sequence on each tag oligonucleotide sequence is used to amplify the nucleic acid molecule. In some embodiments, the primer may be a universal primer or a unique primer sequence. Amplification allows for the preparation of target nucleic acids for sequencing, and the retrieval of target nucleic acids having a desired sequence after sequencing. In some embodiments, a sample of nucleic acid molecules is subjected to transposon-mediated fragmentation and adaptor ligation to enable rapid preparation of paired-end reads using a high-throughput sequencing system. For example, it can be prepared by Nextera^TMLabeled (Hamminda) samples.

One skilled in the art will appreciate the importance of controlling the degree of fragmentation and the size of the nucleic acid fragments to maximize the number of reads in a sequencing paired read to enable sequencing of the desired length of the fragments. In some embodiments, paired end reads can result in one sequence with a tag for identification, and a sequence within the target region of another construct. With high throughput sequencing, sufficient coverage can be generated to reconstruct the consensus sequence of each tag pair construct and determine if the construct sequence is correct. In some embodiments, it is preferable to limit the number of disconnections to less than 2, less than 3, or less than 4. In some embodiments, the degree of fragmentation and/or size of the fragments may be controlled using suitable reaction conditions, such as by using a suitable transposase concentration and controlling the temperature and time of incubation. A standard curve of the actual sample library can be established by using known amounts of the test library and titrating the enzyme and time to obtain appropriate reaction conditions. In some embodiments, portions of the sample not used for fragmentation can be remixed with the fragmented sample and processed for sequencing.

The sample can then be sequenced on the platform that generates paired end reads. Depending on the size of the individual DNA constructs, the number of constructs mixed together, and the estimated error rate of the population, a suitable platform can be selected to maximize the number of reads required and minimize the cost per construct.

Sequencing of nucleic acid molecules results in the reading of two tags from each molecule in paired end reads. Paired end reads can be used to identify which tag pair is ligated or PCR-bound and the identity of the molecule.

Data analysis

In some embodiments, sequencing data or reads are analyzed according to the scheme of fig. 5. Reads may indicate consecutive base calls associated with the sequence of the nucleic acid. It will be understood that reads may include the full length sequence of the sample nucleic acid template or portions thereof, such as sequences comprising barcode sequences, sequence identifiers and portions of the target sequence. Reading may comprise a small number of base calls, such as about 8 nucleotides (base calls) but also contain a larger number of base calls, such as 16 or more base calls, 25 or more base calls, 50 or more base calls, 100 or more base calls, or 200 or more nucleotide or base calls.

For data analysis, reads where one tag is paired with multiple other tags of the same construct are discarded, as this would lead to ambiguity for the clone from which the data originated.

The sequencing results can then be analyzed to determine the sequence of each clone of each construct. For each pair of reads, one of which contains a tag sequence, the identity of the molecule from which each sequencing read is derived is known, and the construct sequence itself can be used to distinguish constructs with the same tag. Other reads from paired reads can be used to construct a consensus sequence for the internal region of the molecule. From these results, a map of the tag pairs corresponding to the correct target sequences for each construct can be generated.

According to one embodiment, the analysis may include one or more of the following: (1) feature annotation; (2) correcting the characteristics; (3) identity assignment and confidence; (4) consensus predicate and confidence; and (5) preparative isolation.

Aspects of the invention provide the ability to generate consensus sequences for each nucleic acid construct. Each base call in the sequence can be based on a common base call at a particular position based on multiple reads at that position. These multiple reads are then assembled or compared to provide a consensus determination of a given base at a given position, and thus a consensus sequence for a particular sequence construct. It is to be understood that any method of assigning a consensus determination of a particular base call from multiple reads of that position of the sequence is contemplated and encompassed by the present invention. Methods of determining such determinations are known in the art. Such methods may include a heuristic approach to multiple sequence alignment, an optimal approach to multiple sequence alignment, or any method known in the art. In some embodiments, the sequence reads are compared to a reference sequence (e.g., a predetermined sequence of interest). High throughput sequencing requires efficient algorithms for mapping multiple query sequences, such as short reads of sequence identifiers or barcodes to such reference sequences.

According to some aspects of the invention, feature annotation includes finding primary features and secondary features. For example, alignment using two reads of the sequence identifier SeqID in the pair of reads can filter out constructs that do not have the correct identifier at the 5 'end and 3' end of the construct or the correct barcode sequence at the 5 'end and 3' end of the sequence identifier. In some embodiments, the Levenshtein distance may be used to cluster clones to correct features. Clones can then be ranked based on confidence in the identity assignments.

Isolation of target nucleic acid sequences

Aspects of the invention are particularly useful for isolating a nucleic acid sequence of interest from a collection comprising nucleic acid sequences containing sequence errors. The techniques provided herein can include any non-destructive sequencing method. Non-limiting examples of non-destructive sequencing include pyrosequencing, as originally described by Hyman et al (1988, anal. biochem. 74: 324-436), and bead-based sequencing, as described by, for example, Leamon et al (2004, electrophosphoresis 24: 3769-3777). Non-destructive sequencing also includes methods using cleavable labeled oligonucleotides, as described by Mitra et al (2003, anal. biochem. 320: 55-62), and photocleavable linkers (Seo et al, 2005, PNAS 102: 5926-. The techniques provided herein also include methods using reversible terminators (Metzker et al, 1994, NAR 22: 4259-4267). Other methods of non-destructive sequencing, including single molecule sequencing, are described in U.S. Pat. Nos. 7,133,782 and 7,169,560, which are incorporated herein by reference.

Provided herein are methods for selectively extracting or isolating the correct sequence from an erroneous sequence. The term "selective separation" as used herein may include physical separation of a desired nucleic acid molecule from other nucleic acid molecules by selective physical movement of the desired nucleic acid molecule, selective inactivation, disruption, release or removal of other nucleic acid molecules than the nucleic acid molecule of interest. It will be appreciated that the library of nucleic acid molecules or nucleic acid constructs may include some errors, which may be due to sequence errors introduced during oligonucleotide synthesis, synthesis of the assembled nucleic acids, and/or due to assembly errors during the assembly reaction. Unwanted nucleic acids may be present in some embodiments. For example, 0% to 50% (e.g., less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1%) of the sequences in the library may be unwanted sequences.

In some embodiments, the method for recovering the annotated correct target sequence described herein can be used to recover a target having a desired sequence. In some embodiments, a pair of tag sequences for each correct target sequence may be used to amplify constructs from a sample set by PCR (as shown in fig. 1C, step IV). It should be noted that since the probability of using the same pair for multiple molecules is extremely low, the probability of isolating a nucleic acid molecule with the correct sequence is high. In other embodiments, nucleic acids having a desired sequence can be recovered directly from a sequencer. In some embodiments, once the tag pairs are identified, the identity of the full-length construct can be determined. In principle, the position of the full-length reads (corresponding to paired-end reads containing 5 'and 3' tags) can be determined on the original sequencing flow cell. After the clusters are positioned on the flow cell surface, the molecules can be eluted or captured from the surface.

In some embodiments, the nucleic acid can be sequenced in a sequencing channel. In some embodiments, nucleic acid constructs can be sequenced in situ using solid supports for gene synthesis and reuse/recycle derived therefrom. Analysis of sequence information from oligonucleotides allows identification of those nucleic acid molecules that appear to have the desired sequence and those that do not. Such sequence information analysis may be qualitative, e.g., providing a positive or negative answer as to the presence of one or more sequences of interest (e.g., in an extension of 10 to 120 nucleotides). In some embodiments, the target nucleic acid molecule of interest can then be selectively isolated from the remainder of the population. Sorting of individual nucleic acid molecules can be facilitated by the use of one or more solid supports (e.g., beads, insoluble polymeric materials, flat surfaces, membranes, porous or non-porous surfaces, chips, or any suitable support, etc.) that can immobilize the nucleic acid molecules. For example, the nucleic acid molecule may be immobilized on a porous surface, such as a glass surface or glass beads. In other examples, the nucleic acid can be immobilized on a flow-through system, such as a porous membrane or the like. Nucleic acid molecules determined to have the correct desired sequence can be selectively released or selectively replicated.

If the nucleic acid molecules are located at different positions, for example, in separate wells of a substrate, the nucleic acid molecules can be selectively extracted from wells identified as containing nucleic acid molecules having the desired sequence. For example, in the Margulies et al apparatus, polymerase clone beads are located in separate wells of a fiber optic slide. Physical extraction of the beads from the appropriate wells of the device allows subsequent amplification or purification of the desired nucleic acid molecule free of other contaminating nucleic acid molecules. Alternatively, if the nucleic acid molecule is attached to a bead using a selectively cleavable linker, cleavage of the linker (e.g., by increasing the pH in the well to cleave the base labile linker) can be used followed by extraction of the solvent in the well to selectively separate the nucleic acid molecule without physical manipulation of the bead. Similarly, if the method of sheddere et al is used, physical extraction of the portion of the bead or gel containing the nucleic acid molecule of interest can be used to selectively isolate the desired nucleic acid molecule.

Certain other methods of selective separation include targeting nucleic acid molecules without the need for physical manipulation of the solid support. Such methods may comprise the use of optical systems to specifically target radiation to individual nucleic acid molecules. In some embodiments, the damaging radiation can selectively target unwanted nucleic acid molecules (e.g., using micro-mirror technology) to destroy them or inactivate them, leaving an enriched population of desired nucleic acid molecules. The enriched population may then be released from the solid support and/or amplified (e.g., by PCR).

Examples of methods and systems for selectively separating desired products (e.g., nucleic acids of interest) may use laser tweezers or optical tweezers. Laser tweezers have been used in the fields of biotechnology, medicine and molecular biology for about 20 years to position and manipulate micron-sized and sub-micron-sized particles (a. ashkin, Science, (210), p.1081-1088, 1980). By focusing the laser beam at a desired location (e.g., bead, well, etc.) containing the desired nucleic acid molecule of interest, the desired container remains optically captured while the undesired nucleic acid sequence is eluted. Once all unwanted material is washed away, the optical tweezers can be turned off to release the desired nucleic acid molecules.

Another method of capturing the desired product is by melting away the undesired nucleic acid. In some embodiments, a high power laser may be used to generate sufficient energy to cause nucleic acid molecules in regions where unwanted material is present to be deactivated, degraded, or destroyed. The region where the desired nucleic acid is present is not subjected to any damaging energy and thus retains its contents.

In some embodiments, error-containing nucleic acid constructs can be eliminated. According to some embodiments, the method comprises generating a nucleic acid having oligonucleotide tags at its 5 'end and 3' end. For example, after the target sequence (e.g., full-length nucleic acid construct) is assembled, the target sequence may be barcoded or the target sequence may be assembled from a plurality of oligonucleotides designed to have barcodes at the 5 'end and 3' end of the target sequence. Tagged target sequences can be fragmented and sequenced using, for example, next generation sequencing as described herein. After the error-free target sequences are identified, the error-free target sequences can be recovered directly from the next generation sequencing plate. In some embodiments, the elimination of error-containing nucleic acids may be accomplished using laser ablation or any suitable method capable of eliminating unwanted nucleic acid sequences. Error-free nucleic acid sequences can be eluted from the sequencing plate. Primers specific to the target sequence can be used to amplify the eluted nucleic acid sequence.

In some embodiments, the target polynucleotide may be amplified after the clonal population is obtained. In some embodiments, the target polynucleotide may comprise a universal (common to all oligonucleotides), semi-universal (common to at least a portion of the oligonucleotides), or individual or unique primer (specific for each oligonucleotide) binding site on the 5 'end or the 3' end or both ends. The term "universal" primer or primer binding site as used herein means that the sequence used to amplify the oligonucleotide is common to all oligonucleotides, so that all such oligonucleotides can be amplified using a single set of universal primers. In other circumstances, the oligonucleotide comprises a unique primer binding site. The term "unique primer binding site" as used herein refers to a set of primer recognition sequences that selectively amplify a subset of oligonucleotides. In other circumstances, the target nucleic acid comprises universal and unique amplification sequences, which may optionally be used sequentially.

In some aspects of the invention, a binding tag capable of binding an error-free nucleic acid molecule or a solid support comprising a binding tag can be added to an error-free nucleic acid sequence. For example, a binding tag, a solid support comprising a binding tag, or a solid support capable of binding nucleic acids can be added to a location on a sequencing plate or flow cell that is identified as comprising an error-free nucleic acid sequence. In some embodiments, the binding tag has a sequence complementary to a target nucleic acid sequence. In some embodiments, the binding tag is a double-stranded sequence designed to hybridize or ligate capture a nucleic acid of interest.

In some embodiments, the solid support may be a bead. In some embodiments, the beads may be deposited on a substrate. The beads can be deposited on the substrate in a variety of ways. The beads or particles may be deposited on a surface of a substrate such as a well or flow cell and may be exposed to a variety of reagents and conditions that allow for the detection of the label or marker. In some embodiments, the sequencing plate can be by ink-jet deposition of binding tags or beads on specific positions.

In some embodiments, the beads can be derived in situ with binding tags complementary to barcodes or additional sequences adsorbed to nucleic acids to capture, and/or enrich for, and/or amplify target nucleic acids identified as having the correct nucleic acid sequence (e.g., error-free nucleic acids). Nucleic acids can be immobilized on the beads by hybridization, covalent attachment, magnetic attachment, affinity attachment, and the like. Hybridization is usually performed under stringent conditions. In some embodiments, the binding tag may be a universal or common primer that is complementary to a non-target sequence (e.g., a full barcode) or an adsorbed additional sequence. In some embodiments, each bead may have a binding tag capable of binding to sequences present at the 5 'end and 3' end of the target molecule. Upon binding of the target molecule, a circular binding is generated. In other embodiments, the beads may have a binding tag capable of binding to a sequence present at the 3' end of the target molecule. In other embodiments, the beads may have a binding tag capable of binding to a sequence present at the 5' end of the target molecule.

Beads, such as magnetic or paramagnetic beads, can be added to each well or arrayed on a solid support. For example, Solid Phase Reversible Immobilization (SPRI) beads from Beckman Coulter (Beckman Coulter) may be used. In some embodiments, the collection of constructs can be distributed in separate wells containing beads. Additional thermal cycling may be used to enhance capture specificity. Using standard magnetic capture, the solution can then be removed, followed by washing of the coupled beads. The desired construct clones may be amplified on beads or after release of the captured clones. In some embodiments, the beads can be configured for hybridization or ligation based capture using double stranded sequences on the beads.

Various bead-based methods may include a set of flowably sorted encoded beads. Bead-based methods may employ nucleic acids hybridized to capture probes or attached to the surface of different populations of capture beads. Such encoded beads can be used on a collection of constructs, then sorted into individual wells for downstream amplification, isolation, and cleaning. Although the magnetic bead use described above may be particularly useful, other methods of separating beads are contemplated in some aspects of the invention. The capture beads can be labeled with a fluorescent moiety that allows the target capture beads to complex with the fluorescence. For example, the beads may be impregnated with fluorophores to produce different populations of beads, which may be sorted by fluorescence wavelength. The target capture bead complexes can be isolated by flow cytometry or fluorescent cell sorting. In other embodiments, the beads may vary in size, or in any suitable characteristic, to enable sorting of different bead populations. For example, the use of capture beads having different sizes would enable separation by filtration or other particle size separation techniques.

In some embodiments, flowable sort-encoded beads can be used to isolate nucleic acid constructs before or after post-synthesis release. Such procedures allow sorting by construct size, user, etc.

FIG. 11 shows a schematic of a non-limiting exemplary bead-based recovery process. In some embodiments, can be in general beads, such as magnetic beads loaded with primers. Each bead may be derivatized multiple times to allow multiple primers to bind to it. In some embodiments, the derivatization results in two or more different bound primers per bead, or the same bound primer per bead. Such beads may be distributed in each well of a multiwell plate. The beads can be loaded with a barcode capable of capturing a particular nucleic acid molecule, for example by hybridizing a nucleic acid sequence comprising the barcode to a sequence complementary to a primer loaded on a universal bead. The sample comprising the double stranded pooled nucleic acids may be subjected to suitable conditions to render the double stranded nucleic acids single stranded. For example, double-stranded nucleic acids may be subjected to any denaturing conditions known in the art. The pooled single stranded sample may be distributed among all wells of a multiwell plate. Under appropriate conditions, the derivatized beads comprising the barcode can capture a particular nucleic acid molecule in each well based on the exact barcode (K, L) loaded on the bead in each well. The beads can then be washed. For example, when magnetic beads are used, the beads can be pulled down with a magnet to allow washing and removal of the solution. In some embodiments, the beads can be washed repeatedly. PCR can then be used to amplify the nucleic acids remaining bound to the beads to generate individual clones in each well of the multi-well construct plate.

In other aspects of the invention, nanopore sequencing can be used to sequence individual nucleic acid strands at the single nucleotide level. One skilled in the art will appreciate the advantages of nanopore sequencing are the minimization of sample preparation sequencing reads and the absence of nucleotides, polymerases, or ligases, and the potential for very long read lengths. However, nanopore sequencing may have a high error rate (about 10% error per base). In some embodiments, the nano-sequencing device includes a micro-fluidic valve that can be switched to recycle the full-length nucleic acid construct to allow multiple sequencing to occur. In some embodiments, the nanopore may be connected to a series of fluidic valves that can be switched such that the full-length nucleic acid construct can be switched back to the nanopore multiple times to allow multiple sequencing passes. Using these configurations, the full length nucleic acid molecule can be measured two or more times. The resulting error-free nucleic acid sequences can be transferred to collection wells for recovery and use.

In some aspects of the invention, provided herein are alternative preparative sequencing methods. The method includes circularizing a target nucleic acid (e.g., a full length target nucleic acid) using a double-ended primer capable of binding to the 5 'end and the 3' end of the target nucleic acid. In some embodiments, the double-ended primer has a sequence complementary to the 5 'end and 3' end barcodes. Nucleases can be added to degrade the linear nucleic acids, thereby locking in the desired construct. Optionally, primers specific for the target nucleic acid can be used to amplify the target nucleic acid.

Inverted in vitro cloning

In some aspects of the invention, methods are provided for isolating and/or recovering sequence-verified nucleic acids of interest. The methods described herein can be used to recover, e.g., error-free, nucleic acid sequences of interest from a nucleic acid library or collection of nucleic acid sequences. The nucleic acid library or collection of nucleic acid sequences can include one or more target nucleic acid sequences of interest (e.g., N genes). In some embodiments, a library of nucleic acid sequences may comprise constructs assembled from oligonucleotides or nucleic acid fragments. Multiple barcoded constructs can be assembled as described herein. In some embodiments, a barcode library can be used to assemble and barcode multiple constructs, such that each nucleic acid construct can be tagged with a unique barcode at each end. In other embodiments, multiple constructs may be assembled from multiple internal target sequence fragments and unique barcode sequences. For example, a library of nucleic acid sequences may comprise M copies of N different target nucleic acid sequences. For example, a 100 copy of a 96-entry tag sequence and a barcode library may have 316 different barcodes, 100000 combinations. In some embodiments, the barcode library can have the same amplification sequence (e.g., common primer binding sequence) outside of the barcode. In some embodiments, if desired, a common amplification tag can be used to amplify the set of barcoded constructs to have the appropriate nucleic acid concentration for next generation sequencing. In some embodiments, the barcoded constructs may undergo a sequencing reaction from both ends resulting in short paired end reads. In some embodiments and as shown in fig. 13A, barcoded constructs of a set of constructs may be circularized to yield a barcode cohort, independent of the length of the nucleic acid construct. Thus, the amplification of a nucleic acid molecule containing a recognition sequence such as a barcode K_iAnd L_jOr a small nucleic acid fragment of an oligonucleotide tag. Sequencing the recognition sequence to convert K_iAnd L_j(K_i1，L_j1)、(K_i2，L_j2) Etc. For example, sequencing of the recognition sequences can result in C clones having the target sequence based on the identity of their corresponding unique recognition sequence pair. The recognition sequence can then be used to amplify the C-specific source construct molecules in individual wells of a microtiter plate as shown in fig. 13A. For example, if C ═ 8 clones, 8 plates of N entry tag nucleic acid sequences (e.g., 96 genes) can be provided, each plate having a different index tag (fig. 13B). Nextera can be used^TMTagmentation to digest the source molecule (C N) and use

Or higher throughput next generation sequencing platforms to identify the correct target sequence. Sequencing data can be used to identify target nucleic acid sequences and sort sequence-verified nucleic acids of interest. For example, as shown in fig. 13A, well a1 of the left candidate plate may contain sequence-verified nucleic acids of interest. The identified clones can then be recovered from wells that are sufficiently identified to have sequence-verified nucleic acids of interest.

Measuring barcode pair information

In some embodiments, and as shown herein, barcode pairs can be determined by sequencing the full-length molecule. Sequencing from both ends gives the required pairing information. To most efficiently determine barcode pairs using the full-length sequencing method, multiple Nextera's were performed^TMLabeling reaction, in which Nextera^TMThe amount of enzyme varied. These separate reactions can be run in parallel and used while using separate indicesAnd (4) sequencing. The read information can then be combined and processed as a whole. Using such processing schemes allows for the identification of error-free molecules that can subsequently be captured by amplification. However, becauseLength limitation of sequencing (e.g., nuclei over about 1000bpPoor sequencing of acids), barcodes paired using this reaction may not be efficient for constructs over 1000 bp.

According to some embodiments, barcode pair information may be determined according to the method described in fig. 14. FIGS. 14A and 14B illustrate different methods of allowing barcoded ends of molecules to be held together by ligation of blunt ends of constructs into a loop. In both concepts, barcodes can be added to the constructs by PCR using the sequence H1 as a primer site. After dilution and amplification with the H2 primer, the set of constructs can be split into two parallel paths. A portion can be amplified with the H2 primer with the p5 and p7 sequences, p5 and p7 sequences aligned atThe above sequencing is necessary. Can be based on Nextera^TMFragmentation of the amplified construct and subsequent use

And (4) sequencing. The second path focuses on determining barcode pairing information. Referring to fig. 14A, barcode pairs can be amplified and sequenced. Referring to fig. 14B, barcode pairs can be excised from the loop by restriction enzymes and subsequently sequenced. Using the methods described herein, terminal barcode pairs can be linked in a manner that is independent of the length of the sequenced construct.

Figure 14C shows different methods of attaching barcodes to the synthesized constructs. According to some embodiments, compatible nucleic acid overhangs may be opened using restriction enzymes, such as BsaI or any suitable restriction enzyme, which may then be used to ligate paired barcode molecules to the construct, resulting in a circular construct. The collection of circular constructs can then be diluted and amplified with primer H2. The construct is then processed as shown in FIG. 14A or FIG. 14B. FIG. 14D shows a non-limiting embodiment using parallel sequencing of the constructs and isolated barcode pairs to identify the correct molecules for subsequent capture by amplification.

According to some embodiments, the barcode pairs may be divided intoSubsets are generated, each with a single pair of barcodes. Referring to fig. 15, these molecules can be circularized and diluted to the appropriate level, which can be defined by the appropriate total number of barcodes. For example, the number of barcodes may be 10⁵Or 10⁶. The diluted barcodes can then be amplified using multiple displacement amplification to generate multiple copies of each barcode. The resulting bar code collection is then divided in half. The first part can be used to barcode the synthesized construct. The second portion can be sequenced using next generation sequencing. The sequencing data may give barcode-to-barcode correlations within the collection. With appropriate sequencing, completion of the assembly can be determined. It will be appreciated that when such a set is used to sequence constructs, the barcode correlation is known, obviating the need for the process shown in figure 14.

Applications of

Aspects of the invention may be used in a range of applications involving the generation and/or use of synthetic nucleic acids. As described herein, the present invention provides methods of producing synthetic nucleic acids having a desired sequence with improved efficiency. The resulting nucleic acid may be amplified in vitro (e.g., using PCR, LCR, or any suitable amplification technique), amplified in vivo (e.g., by cloning into a suitable vector), isolated, and/or purified. The assembled nucleic acid (alone or cloned into a vector) can be transformed into a host cell (e.g., prokaryotic, eukaryotic, insect, mammalian, or other host cell). In some embodiments, the host cell can be used to propagate the nucleic acid. In certain embodiments, the nucleic acid may be integrated into the genome of the host cell. In some embodiments, the nucleic acid can replace a corresponding nucleic acid region on the genome of the cell (e.g., by homologous recombination). Thus, the nucleic acids may be used to generate recombinant organisms. In some embodiments, the target nucleic acid may be the entire genome or a large fragment of the genome that is used to replace all or part of the genome of the host organism. Recombinant organisms can also be used in a variety of research, industrial, agricultural, and/or medical applications.

Many of the techniques described herein can be used together to generate nucleic acid molecules using suitable assembly techniques at one or more points. For example, ligase-based assembly can be used to assemble oligonucleotide duplexes and nucleic acid fragments of less than 100 to more than 10000 base pairs in length (e.g., 100 mer-500 mer, 500 mer-1000 mer, 1000 mer-5000 mer, 5000 mer-10000 mer, 25000 mer, 50000 mer, 75000 mer, 100000 mer, etc.). In exemplary embodiments, the methods described herein can be used in assembling an entire genome (or large fragments thereof, e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) of an organism (e.g., a virus, bacterium, yeast, or other prokaryotic or eukaryotic organism), optionally incorporating specific modifications into the sequence at one or more desired locations.

Any nucleic acid product (e.g., comprising amplified, cloned, purified, isolated, etc., nucleic acids) can be packaged in any suitable form (e.g., in a stable buffer, lyophilized, etc.) for storage and/or transport (e.g., for transport to a distribution center or user). Similarly, any host cell (e.g., a genomic cell transformed with a vector or having a modification) can be prepared in a suitable buffer for storage and/or transport (e.g., for distribution to a user). In some embodiments, the cells may be frozen. However, other stable cell preparations may also be used.

Host cells can be grown and expanded in culture. Host cells can be used to express one or more RNAs or polypeptides of interest (e.g., therapeutic, industrial, agricultural, and/or pharmaceutical proteins). The expressed polypeptide may be a native polypeptide or a non-native polypeptide. The polypeptide may be isolated or purified for subsequent use.

Thus, a nucleic acid molecule produced using the methods of the invention can be incorporated into a vector. The vector may be a cloning vector or an expression vector. In some embodiments, the vector may be a viral vector. The viral vector may comprise a nucleic acid sequence capable of infecting a target cell. Similarly, in some embodiments, prokaryotic expression vectors operably linked to a suitable promoter system may be used to transform a cell of interest. In other embodiments, eukaryotic vectors operably linked to a suitable promoter system may be used to transfect cells or tissues of interest.

Transcription and/or translation of the constructs described herein can be performed in vitro (e.g., using a cell-free system) or in vivo (e.g., expression in a cell). In some embodiments, cell lysates can be prepared. In certain embodiments, the expressed RNA or polypeptide may be isolated or purified. The nucleic acids of the invention may also be used to add detection and/or purification tags to the expressed polypeptide or fragment thereof. Examples of polypeptide-based fusions/tags include, but are not limited to, hexa-histidine (His)⁶) Myc and HA, and other polypeptides with multiple uses, such as GFP₅GST, MBP, chitin, etc. In some embodiments, a polypeptide may comprise one or more unnatural amino acid residue.

In some embodiments, antibodies can be made against polypeptides encoded by one or more synthetic nucleic acids, or fragments thereof. In certain embodiments, synthetic polypeptides may be provided as a library for screening in research and development (e.g., to identify potential therapeutic proteins or polypeptides, to identify potential protein targets for drug development, etc.). In some embodiments, the synthetic nucleic acids can be used as therapeutics (e.g., for gene therapy, or for gene regulation). For example, the synthetic nucleic acid can be provided to a patient in an amount sufficient to express a therapeutic amount of the protein. In other embodiments, the synthetic nucleic acid can be administered to the patient in an amount sufficient to regulate (e.g., down-regulate) gene expression.

It is to be understood that the different acts or embodiments described herein may be performed independently and may be performed in different locations in or outside of the united states. For example, each act and any other act or any portion of these acts in accepting an order for a target nucleic acid, analyzing a target nucleic acid sequence, designing one or more starting nucleic acids (e.g., oligonucleotides), synthesizing starting nucleic acids, purifying starting nucleic acids, assembling starting nucleic acids, isolating assembled nucleic acids, confirming the sequence of assembled nucleic acids, manipulating assembled nucleic acids (e.g., amplifying, cloning, inserting into a host genome, etc.) can be performed separately at one location or at different locations within or outside the united states. In some embodiments, the assembly process may include a combination of actions performed at one location (within or outside the united states) and at one or more remote locations (within or outside the united states).

Automated applications

The method and apparatus aspects provided herein may include automating one or more actions (acts) described herein. In some embodiments, one or more steps in an amplification and/or assembly reaction may be automated using one or more automated sample processing devices (e.g., one or more automated liquid or fluid processing apparatus). Automated apparatus and methods may be used to deliver a reaction reagent, comprising one or more of the following: starting nucleic acids, buffers, enzymes (such as one or more ligases and/or polymerases), nucleotides, salts, and any other reagents such as stabilizers. Automated devices and methods may also be used to control reaction conditions. For example, an automated thermal cycler can be used to control the reaction temperature and any reaction cycles that can be used. In some embodiments, the scanning laser may be automated to provide one or more reaction temperatures or temperature cycles suitable for incubating the polynucleotide. Similarly, subsequent analysis of the assembled polynucleotide product can be automated. For example, sequencing can be automated using sequencing equipment and automated sequencing protocols. Other steps (e.g., amplification, cloning, etc.) can also be automated using one or more suitable devices and associated protocols. It should be understood that one or more of the devices or device components described herein may be combined in a system (e.g., a robotic system) or in a micro-environment (e.g., a microfluidic reaction chamber). Assembly reaction mixtures (e.g., liquid reaction samples) can be transferred from one component of the system to another using automated equipment and methods (e.g., mechanized handling and/or transfer of samples and/or sample containers, including automated pipetting equipment, microsystems, etc.). The system and any components thereof may be controlled by a control system.

Thus, the method steps and/or content of the apparatus provided herein can be automated using, for example, a computer system (e.g., a computer control system). A computer system capable of implementing aspects of the techniques provided herein may comprise a computer for any type of process (e.g., sequence analysis and/or automated device control as described herein). However, it should be understood that certain process steps may be provided by one or more automated devices that are part of the assembly system. In some embodiments, a computer system may include two or more computers. For example, one computer may be coupled to a second computer via a network. A computer may perform the sequence analysis. The second computer may control one or more automated compounding and assembly devices in the system. In other aspects, other computers may be included in the network to control one or more of the analysis or processing actions. Each computer may include a memory and a processor. The computer may take any form, as the techniques provided herein are not limited to implementation on any particular computer platform. Similarly, the network can take any form, including a private network or a public network (e.g., the Internet). The display device can be associated with one or more devices and computers. Alternatively, or in addition, a display device may be located at a distal site and connected to display the analysis output according to the techniques provided herein. The connections between the various components of the system may be via wire, fiber optic, wireless transmission, satellite transmission, any other suitable transmission, or any combination of two or more of the foregoing.

Various aspects, embodiments, or functions of the technology provided herein can be independently automated and performed in a variety of ways. For example, the various aspects, embodiments or functions can be implemented independently using hardware, software, or a combination thereof. When implemented in software, the software code can be executed on any suitable processor or collection of processors, whether provided in a single computer or distributed across multiple computers. It should be appreciated that any component or collection of components that perform the functions described above can be generically considered as one or more controllers that control the functions discussed above. The one or more controllers can be implemented in numerous ways, such as with dedicated hardware or with general purpose hardware (e.g., one or more processors) that is controlled using microcode or software routines to perform the functions recited above.

In this regard, it will be appreciated that one implementation of the techniques provided herein comprises at least one computer-readable medium (e.g., a computer memory, a floppy disk, a compact disk, a tape, etc.) encoded with a computer program (e.g., with a plurality of instructions), which, when executed on a processor, performs one or more of the functions described above for the techniques provided herein. The computer readable medium is transportable, such that the program stored thereon can be loaded onto any computer system source to perform one or more functions of the techniques provided herein. Further, it should be understood that references to a computer program that performs the functions discussed above, when executed, are not limited to running an application on a host computer. Rather, the term computer program is used herein in a generic sense to refer to any type of computer code (e.g., software or microcode) that can be employed to program a processor to perform the aspects of the techniques provided herein discussed above.

It should be understood that consistent with several embodiments of the techniques provided herein where a processor is stored on a computer-readable medium, the computer-implemented process may receive manual input (e.g., from a user) during execution thereof.

Thus, overall system level control of the assembly devices or assemblies described herein may be performed by a system controller, which may provide control signals to: related nucleic acid synthesizers, liquid handling equipment, thermocyclers, sequencing equipment, related mechanized components, and other suitable systems for performing the desired input/output or other control functions. Thus, the system controller, together with any device controller, forms a controller that controls the operation of the nucleic acid assembly system. The controller may include a general purpose data processing system, which may be a general purpose computer or a network of general purpose computers, and other related devices including communication devices, modems, and/or other circuits or components to perform the required input/output or other functions. The controller can also be implemented at least in part as a single special purpose integrated circuit (e.g., ASIC) or an ASIC array, each having a main or central processor section for overall, system-level control, and a dedicated, discrete section for performing a variety of different specific computations, functions, and other processes under the control of the central processor section. The controller can also be implemented using a variety of separate application specific program integration or other electronic circuits or devices, such as hardwired electronic devices or logic circuits, e.g., separate component circuits or program logic devices. The controller can also include any other components or devices, such as user input/output devices (monitors, displays, printers, keyboards, user pointing devices, touch screens, or other user interfaces, etc.), data storage devices, drive motors, connections, valve controllers, motorized devices, vacuum and other pumps, pressure sensors, detectors, power supplies, pulse sources, communication devices or other electronic circuits or components, and so forth. The controller may also control the operation of other parts of the system, such as automated customer order processing, quality control, packaging, shipping, invoicing, etc., to perform other suitable functions known in the art and not described in detail herein.

Various aspects of the present invention may be used alone, in combination, or in a variety of arrangements not specifically discussed in the embodiments described in the foregoing and is therefore not limited in its application to the details and arrangement of components set forth in the foregoing description or illustrated in the drawings. For example, the aspects of one embodiment may be combined in any manner with the aspects of the other embodiments.

The use of ordinal terms "first," "second," "third," etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

Also, the phraseology and terminology used herein is for the purpose of description and not of limitation. The use of "including," "comprising," or "having," "containing," "involving," and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

The following examples are set forth as representative of the invention. These examples are not to be construed as limiting the scope of the invention thereto and other equivalent embodiments will be apparent from the disclosure, drawings and appended claims.

Examples